EP2210612B1 - Macrocyclic antagonists of the motilin receptor - Google Patents
Macrocyclic antagonists of the motilin receptor Download PDFInfo
- Publication number
- EP2210612B1 EP2210612B1 EP10001568.4A EP10001568A EP2210612B1 EP 2210612 B1 EP2210612 B1 EP 2210612B1 EP 10001568 A EP10001568 A EP 10001568A EP 2210612 B1 EP2210612 B1 EP 2210612B1
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- EP
- European Patent Office
- Prior art keywords
- boc
- compound
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- Prior art date
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- 108700040483 Motilin receptors Proteins 0.000 title description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 109
- 238000000034 method Methods 0.000 claims abstract description 65
- 150000002678 macrocyclic compounds Chemical class 0.000 claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
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- 125000006242 amine protecting group Chemical group 0.000 claims description 4
- 238000009903 catalytic hydrogenation reaction Methods 0.000 claims description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 3
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
- C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
- C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D498/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
Definitions
- the present invention relates to novel conformationally-defined macrocyclic compounds, pharmaceutical compositions comprising same and intermediates used in their manufacture. More particularly, the invention relates to macrocyclic compounds that have been demonstrated to selectively antagonize the activity of the motilin receptor. The invention further relates to macrocyclic compounds useful as therapeutics for a range of gastrointestinal disorders, in particular those in which malfunction of gastric motility or increased motilin secretion is observed, such as hypermotilinemia, irritable bowel syndrome and dyspepsia.
- GI gastrointestinal
- absorption, secretion, blood flow and motility Mulvihill, et al. in Basic and Clinical Endocrinology, 4th edition, Greenspan, F.S.; Baxter, J.D., eds., Appleton & Lange: Norwalk, CT, 1994, pp 551-570 ). Since interactions between the brain and GI system are critical to the proper modulation of these functions, these peptides can be produced locally in the GI tract or distally in the CNS.
- motilin a linear 22-amino acid peptide
- motilin plays a critical regulatory role in the CI physiological system though governing of fasting gastrointestinal motor activity.
- the peptide is periodically released from the duodenal mucosa during fasting In mammals, including humans. More precisely, motilin exerts a powerful effect on gastric mobility through the contraction of gastrointestinal smooth muscle to stimulate gastric emptying, decrease Intestinal transit time and initiate phase III of the migrating motor complex In the small bowel ( Itoh, Z., Ed., Motilin, Academic Press: San Diego, CA, 1990 , ASIN: 0123757304; Nelson, D.K. Dig. Dis. Sci. 1996, 41, 2006-2015 ; Peeters, T.L.; Vantrappen, G.; Janssens, J. Gastroenterology 1980, 79, 716-719 ).
- Motilin exerts these effects through receptors located predominantly on the human antrum and proximal duodenum, although its receptors are found in other regions of the GI tract as well ( Peeters, T.L.; Bormans, V.; Vantrappen, G. Regul. Pept 1989, 23, 71-182 ). Therefore, motilin hormone is involved In mobility of both the upper and lower parts of the GI system ( williams et al. Am. J. Physiol.1992, 262, G50-G55 ).
- motilin and its receptors have been found In the CNS and periphery, suggesting a physiological role in the nervous system that has not yet been definitively elucidated ( Depoortere, I.; Peeters, T.L. Am. J. Physiol. 1997, 272, G994-999 and O'Donohue, T.L et al. Peptides 1981, 2, 487.477 ).
- motilin receptors In the brain have been suggested to play a regulatory role in a number of CNS functions, including feeding and drinking behavior, micturition reflex, central and brain stem neuronal modulation and pituitary hormone secretion ( Itoh, Z. Motilln and Clinical Applications.
- motilin Due to the critical and direct involvement of motilin in control of gastric motility, agents that either diminish (hypomotility) or enhance (hypermotility) the activity at the motilin receptor, are a particularly attractive area for further investigation in the search for new effective pharmaceuticals towards these indications.
- Peptidlc agonists of the motilin receptor which have clinical application for the treatment of hypomotility disorders, have been reported ( U.S. 5,695,952 ; 5,721,353 ; 6,018,037 ; 6,380,158 ; 6,420,521 , U.S. Appl. 2001/0041791 , WO 98/42840 ; WO 01/00830 and WO 02/059141 ).
- Derivatives of erythromycin commonly referred to as motilides, have also been reported as agonists of the motilin receptor ( U.S.
- Antagonists of the motilin receptor are potentially extremely useful as therapeutic treatments for diseases associated with hypermotility and hypermotilinemia, including irritable bowel syndrome, dyspepsia, gastroesophogeal reflux disorders, Crohn's disease, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, diabetes mellitus, obesity, malabsorption syndrome, carcinoid syndrome, diarrhea, atrophic colitis or gastritis, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, gastric stasis and eating disorders leading to obesity.
- diseases associated with hypermotility and hypermotilinemia including irritable bowel syndrome, dyspepsia, gastroesophogeal reflux disorders, Crohn's disease, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, diabetes mellitus, obesity, malabsorption syndrome, carcinoid syndrome, diarrhea, atrophic colitis or gastritis,
- peptidic compounds have been described as antagonists of the motilin receptor ( Depoortere, I.; Macielag, M.J.; Galdes, A.; Peeters, T. L. Eur. J. Pharmacol. 1995, 286, 241-247 ; US 5,470,830 ; 6,255,285 ; 6,586,630 ; 6,720,433 ; U.S. 2003/0176643 ; WO 02/64623 ).
- These peptidic antagonists suffer from the known limitations of peptides as drug molecules, in particular poor oral bioavailability and degradative metabolism.
- Cyclization of peptidic derivatives is a method employed to improve the properties of a linear peptide both with respect to metabolic stability and conformational freedom. Cyclic molecules tend to be more resistant to metabolic enzymes. Such cyclic tetrapeptide motilin antagonists have been reported ( Haramura, M. et al J. Med. Chem. 2002, 45, 670-675 , U.S. 2003/0191053 ; WO 02/16404 ).
- the macrocyclic motilin antagonists of the present invention comprise elements of both peptidic and non-peptidic structures in a combination which has not been pursued for this application previously.
- antagonists of the present invention are different.
- the known motilin antagonists which are cyclic peptides it was found that such derivatives containing D-amino acids were devoid of activity.
- the tripeptidomimetic compounds of the present invention the D-stereochemistry is required for two of the three building elements.
- the motilin antagonists of the present invention are also distinct from the prior art in that they comprise a tether element to fulfill the dual role of controlling conformations and providing additional sites for interaction either through hydrophobic interactions, hydrogen bonding or dipole-dipole interactions.
- the present invention provides a conformationally-defined tripepeptidomimetic macrocyclic compound as defined in claim 1.
- the conformationally-defined tripepeptidomimetic macrocyclic compounds of the invention may be of formula (I) and pharmaceutical acceptable salts, hydrates or solvates thereof wherein:
- the invention provides a process as defined in claim 5.
- the invention provides a compound as defined in claim 7.
- the invention provides a method as defined in claim 11.
- R 1 is selected from the group consisting of -(CH 2 ) q R 11 , and -CHR 12 R 13 wherein q is 0, 1, 2 or 3; and R 11 and R 12 are independently selected from a ring structure from the following group: wherein any carbon atom in said ring structure can be replaced a nitrogen atom, with the proviso that if said ring structure is a monocyclic ring structure, It does not comprise more than four nitrogen atoms and If said ring structure is a bicyclic ring structure, it does not comprise more than six nitrogen atoms:
- R 11 , R 12 and R 13 are selected from the group consisting of: wherein R a and R b are chosen from the group consisting of Cl, F, CF 3 , OCH 3 , OH, and C(CH 3 ) 3 and CH 3 .
- the tether portion (T) of formula (I) is preferably selected from the group consisting of:
- compounds of formula (I), wherein m, n and p are 0, X, Z 1 , Z 2 and Z 3 are NH and R 2 , R 4 and R 5 are hydrogen, are represented by formula (III):
- Compounds of formula (I), wherein when Z 1 is a nitrogen atom, R 3 forms a four, five, six or seven-membered heterocyclic ring together with Z 1 are represented by formula (IV): wherein said heterocyclic ring may contain a second nitrogen atom, or an oxygen, or sulfur atom;
- n 2 is selected from 0, 1, 2 or 3
- R 7 is optionally present and is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl,
- amino, guanidine, ureido and amidino encompass substituted derivatives thereof as well.
- Also described herein is a method of treating a disorder associated with hypermotility or hypermotilinemia in humans and other mammals comprising administering a therapeutically effective amount of a compound of formula (I).
- PG and PG' indicate a standard amine protecting group compatible with the synthetic protocol , such as Boc , Ddz, Fmoc, or Alloc
- Also described herein is a method of treating Irritable bowel syndrome, dyspepsia, Crohn's disease, gastroesophogeal reflux disorders, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric, stenosis, carcinoid syndrome, matabsorption syndrome, diarrhea, diabetes mellitus, obesity, postgastroenterectomy syndrome, atrophic colitis or gastritis, gastric stasis, gastrointestinal dumping syndrome, celiac disease and eating disorders leading to obesity in humans and other mammals comprising administering a therapeutically effective amount of a compound of formula (I).
- Reagents and solvents were of reagent quality or better and were used as obtained from various commercial suppliers unless otherwise noted.
- DMF, DCM and THF used are of DriSolv ® (EM Science, E. Merck) or synthesis grade quality except for (i) deprotection, (ii) resin capping reactions and (iii) washing.
- NMP used for the amino add (AA) coupling reactions is of analytical grade.
- DMF was adequately degassed by placing under vacuum for a minimum of 30 min prior to use.
- Tyr(3tBu) was synthesized following the method reported in JP2000 44595 . Cpa was made using literature methods ( Tetrahedron: Asymmetry 2003, 14, 3575-3580 ) or obtained commercially.
- Boc- and Fmoc-protected amino acids and side chain protected derivatives were obtained from commercial suppliers or synthesized through standard methodologies known to those in the art.
- Ddz-amino acids were either synthesized by standard procedures or obtained commercially from Orpegen (Heidelberg, Germany) or Advanced ChemTech (Louisville, KY, USA).
- Bts-amino acids were synthesized as described in Example 6. Hydroxy acids were obtained from commercial suppliers or synthesized from the corresponding amino acids by literature methods.
- Analytical TLC was performed on pre-coated plates of silica gel 60F254 (0.25 mm thickness) containing a fluorescent Indicator.
- concentrated/evaporated under reduced pressure indicates evaporation utilizing a rotary evaporator under either water aspirator pressure or the stronger vacuum provided by a mechanical oil vacuum pump as appropriate for the solvent being removed.
- “Dry pack” indicates chromatography on silica gel that has not been pre-treated with solvent, generally applied on larger scales for purifications where a large difference in R f exists between the desired product and any impurities.
- "dried In the standard manner” Is that the resin is dried first in air (1 h), and subsequently under vacuum (oil pump usually) until full dryness is attained ( ⁇ 30 min to O/N).
- the reaction mixture was washed with Et 2 O. Washing is continued until the absence of non-polar impurities In the aqueous layer Is confirmed by TLC (typically 3 x 100 mL).
- the aqueous solution was then cooled to 0°C, acidified to pH 2.0 with 1 N HCl until no additional cloudiness forms, and extracted with EtOAc (3 x 100 mL).
- EtOAc 3 x 100 mL
- a mixture of DCM and EtOAc may be used as the extraction solvent, depending on the solubility of the product obtained from different amino adds or derivatives. Note that DCM cannot be used solely as solvent because of the emulsion formed during extraction.
- the compounds of Formula I can be synthesized using traditional solution synthesis techniques or solid phase chemistry methods. In either, the construction involves four phases: first, synthesis of the building blocks, including one to four moieties, comprising recognition elements for the biological target receptor, plus one tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations. The precursors from the assembly are then cyclized in the third stage to provide the macrocyclic structures. Finally, a post-cyclization processing stage involving removal of protecting groups and optional purification then provides the desired final compounds (Scheme 1). This method has been previously disclosed in WO 01/25257 and U.S. Pat. Appl. Ser. No. 09/679,331 .
- Tethers T8, T10, T12, T28, T34, T41 and T42 are not compounds of the invention.
- Step T8-1 Chlorotrimethylsilane (116 mL, 0.91 mol, 1.5 eq) was added to a suspension of 2-hydroxycinnamic acid (100 g, 0.61 mol, 1.0 eq) in MeOH (500 mL, HPLC grade) over 30 min at 0° C. The resulting mixture was stirred at rt O/N. The reaction was monitored by TLC (EtOAc/MeOH: 98/2). Heating the reaction mixture in a hot water can accelerate the process if necessary. After the reaction was completed, the reaction mixture was evaporated under reduced pressure to afford methyl 2-hydroxycinnamate as a white solid (108.5 g) in quantitative yield. The identity of this intermediate compound is confirmed by NMR. This reaction can be carried out on larger (kg) scale with similar results
- Step T8-2 3,4-Dihydro-2 H -pyran (DHP, 140 mL, 1.54 mol, 2.52 eq) was added dropwise to 2-bromoethanol (108 mL, 1.51 mol, 2.5 eq) In a 2 L three-neck flask with mechanical stirring at 0° C over 2 h. The resulting mixture was stirred for additional 1 h at rt.
- Methyl 2-hydroxycinnamate from Step T8-1 (108 g, 0.61 mol, 1.0 eq), potassium carbonate (92.2 g, 0.67 mol,1.1 eq).
- StepT8-3 DIBAL (1.525 L, 1.525 mol, 2.5 eq, 1.0 M in DCM) was added slowly to a solution of the above crude ester from Step T8-2 (0.61 mol based on the theoretical yield) In anhydrous DCM (610 mL) at -35° C with mechanical stirring over 1.5 h. The resulting mixture was stirred for 1.5 h at -35° C, then 1.5 h at 0° C. The reaction was monitored by TLC (hextEtOAc: 50/50). When complete, Na 2 SO 4 ⁇ 10 H 2 O (100 g, 0.5 eq) was slowly added; hydrogen evolution was observed, when it subsided water was added (100 mL).
- the mixture was warmed to rt and stirred for 10 min, then warmed to 40° C with hot water and stirred under reflux for 20 min.
- the mixture was cooled to rt, diluted with DCM (600 mL), and the upper solution decanted into a filter.
- the solid that remained in the flask was washed with dichloromethane (5 x 500 mL) with mechanical stirring and filtered.
- the filtrate from each wash was checked by TLC, and additional washes performed if necessary to recover additional product.
- the mixture was filtered.
- the resulting solid was then continuously extracted with 0.5% TEA in dichloromethane using a Soxhlet extractor.
- Step TB-4 To a mixture of the allylic alcohol from Step T8-3 (28 g, 0.100 mol, 1.0 eq) and collidine (0.110 mol, 1.1 eq) In 200 mL of anhydrous DMF under N 2 was added anhydrous LICl (4.26 g, 0.100 mol, 1.0 eq.) dissolved in 100 mL of anhydrous DMF. The mixture was then cooled to 0°C, and MsCl (12.67 g, 0.110 mol, 1.1 eq)., freshly distilled over P 2 O 5 ), was added dropwise. The reaction was allowed to warm to rt and monitored by TLC (3:7 EtOAc/hex).
- Step T8-5 PPh 3 (25.9 g, 0.099 mol,1.5 eq) was added at 0° C to a solution of the allylic azide from Step T8-4 (20.0 g, 0.066 mol, 1.0 eq.) in 100 mL of THF. The solution was stirred for 30 min at 0° C and 20 h at rt. Water (12 mL) was then added and the resulting solution was heated at 60° C for 4 h. The solution was cooled to rt, 2N HCl (15 mL) added and the mixture stirred for 90 min at 50°C. The separated organic phase was extracted with 0.05 N HCl (2 x 100 mL).
- the combined aqueous phase was washed with Et 2 O (5 x 150 mL) and toluene (4 x 150 mL) (more extraction could be necessary, follow by TLC), which were combined and back-extracted with 0.05 N HCl (1 x 100 mL).
- This acidic aqueous phase from back-extraction was combined with the main aqueous phase and washed with ether (5 x 150 mL) again.
- the pH of the aqueous phase was then adjusted to 8-9 by the addition of sodium hydroxide (5 N). Care must be exercised to not adjust the pH above 9 due to the reaction conditions required by the next step.
- the aqueous phase was concentrated under reduced pressure (aspirator, then oil pump) or lyophilized to dryness. Toluene (2x) was added to the residue and then also evaporated under reduced pressure to remove traces of water.
- the crude product was used for the next reaction without further purification.
- Step T8-6 A mixture of the crude amino alcohol from Step T8-5 (0.5 mol based on the theoretical yield), Ddz-OPh (174 g, 0.55 mol, 1.1 eq) and Et 3 N (70 mL, 0.5 mol, 1.0 eq) in DMF (180 mL) was stirred for 24 h at 50° C. Additional DMF is added if required to solubilize all materials. The reaction was monitored by TLC (hex/EtOAc: 50/50, ninhydrin detection). After the reaction was complete, the reaction mixture was diluted with Et 2 O (1.5 L) and water (300 mL). The separated aqueous phase was extracted with Et 2 O (2 x 150 mL).
- Tether T9 can also be synthesized from T8 by reduction as in step T9-3 or with other appropriate hydrogenation catalysts known to those In the art.
- the resin was filtered and washed 2x DCM, 1x toluene, 1x EtOH, 1x toluene, 1x (DCM/MeOH), 1x (THF/MeOH), 1x (DCM/MeOH), 1x (THF/MeOH), 2x DCM, then dried in the standard manner.
- the resin was filtered and washed DCM (2x), toluene (1x), EtOH (1x), toluene (1x), DCM/MeOH (1x), 1x THF/MeOH (1x), DCM/MeOH (1x), THF/MeOH (1x), 2x DCM, then dried in the standard manner.
- Example 29 In certain instances, the Mitsunobu process of Example 29 cannot be applied or is not efficient for incorporation of the tether. Hence, reductive amination has been developed as an alternative that can be employed for tether incorporation as Illustrated hereinbelow for one of the preferred tethers. Similar chemistry can be used to Incorporate other tethers of the present invention.
- the Tether (30-2) with the amine protected as its Ddz derivative was efficiently oxidized to the corresponding aldehyde 30-2 using SO 3 ⁇ pyr in DMSO-Et 3 N-DCM.
- This aldehyde (0.14 mmol, 56 mg, 1.5 eq based upon loading of resin support) was dissolved in a 1:3 mixture of TMOF-MeOH (DriSolv, 4 mL) at rt.
- Chiral material can be accessed by starting with the chiral epoxide.
- the (S)-isomer of T38 was constructed in 89% overall yield from (S)-propylene oxide.
- Chiral T39 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature.
- Chiral T40 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature.
- One or more of the amino acids indicated can be replaced by corresponding hydroxy acids and coupled to the next building block utilizing methods known to those in the art.
- the resin containing the cyclization precursor is combined in an appropriate vessel with pre-washed MP-carbonate resin [Argonaut Technologies, Foster City, CA, commercially supplied MP-carbonate resin was treated with 3x THF (1 L per 400 g) and dried O/N at 30°C in a vacuum oven] (1.4 to 1.6 eq relative to the initial loading of the synthesis resin).
- a 0.2 M DIPEA solution in THF was then added to the combined resins (1 mL / 60 mg MP-carbonate resin) and the suspension agitated O/N at rt. Subsequently, the resin was filtered and rinsed 2x THF.
- the combined filtrates are collected together in an appropriate vessel, then the volatile contents evaporated under reduced pressure [in addition to the standard methods, solvent can also be removed in vacuo using centrifugal evaporation (ThermoSavant Discovery, SpeedVac or comparable)] to provide the crude macrocycles.
- Example 47 Except for the cyclization itself and subsequent work-up, this procedure is Identical to that of Example 47.
- the resin containing the cyclization precursor was combined in an appropriate vessel with pre-washed MP-carbonate resin [Argonaut Technologies, commercially supplied MP-carbonate resin was treated with THF (3x, 1 L per 400 g) and dried O/N at 30°C in a vacuum oven] (1.4 to 1.6 eq relative to the initial loading of the synthesis resin). To this was added THF (1 mL per 100 mg resin) and silver trifluoroacetate (1 eq relative to the initial loading of the resin). Finally, an amount of DIPEA sufficient to obtain a 0.2 M solution was added. The reaction mixture was agitated at rt O/N.
- a ring-closing metathesis (RCM) strategy as developed by Grubbs et al. can also be used to access some of the macrocyclic compounds of the invention (see for example US 5,811,515 ; Grubbs, R.H. et al. J. Org. Chem. 2001, 66, 5291-5300 ; Princestner, A. Angew. Chem. Int. Ed. 2000, 39, 3012-3043 ).
- RCM ring-closing metathesis
- the reaction was best performed after formation of the macrocyclic ring.
- further reaction of amino moieties on side chains examples was typically efficiently done by reaction of the partially protected macrocycle.
- acylation, sulfonylation, alkylation (via reductive amination), guanidine and urea formation were performed via standard methods.
- Table 1 hereinbelow, shows a representative, but by no means exclusive, summary of the chemical synthesis of several representative compounds of the invention (compounds 1, 2, 5-9, 11, 12, 20, 21, 34, 38-41, 52, 55-60, 65, 71, 72, 76, 77, 80, 85, 98, 99, 120, 121, 127, 134, 177, 194, 197 and 199-202 not being compounds of the invention).
- HPLC analyses are performed on a Waters Alliance system 2695 running at 1 mL/min using an Xterra MS C18 column 4.6 x 50 mm (3. 5 ⁇ m).
- a Waters 996 PDA provided UV data for purity assessment.
- An LCPackings splitter (50:40:10) allowed the flow to be separated in three parts. The first part (50%) went to a Micromass Platform II MS equipped with an APCI probe for identity confirmation. The second part (40%) went to an evaporative light scattering detector (ELSD, Polymer Laboratories, PL-ELS-1000) for purity assessment and the last portion (10%) to a chemiluminescence nitrogen detector (CLND, Antek Model 8060) for quantitatlon and purity assessment. Data was captured and processed utilising the most recent version of the Waters Millenium software package.
- ELSD evaporative light scattering detector
- CLND chemiluminescence nitrogen detector
- An example LC method suitable for compounds of the present invention uses MeOH as solvent A, H 2 O as solvent B and 1%TFA/ H 2 O as solvent D.
- Initial mobile-phase composition is 5% A, 85% B and 10% D. Details of the standard gradient method are shown below: Time A% B% D% Curve 0.00 5 85 10 6 1.00 5 85 10 6 6.00 50 40 10 6 9.00 50 40 10 6 14.00 90 0 10 6 17.00 90 0 10 6 17.50 5 85 10 6 20.00 5 85 10 6
- Table 2 Analysis of selected compounds of the invention (Compounds 1, 2, 5-12, 20, 21, 34, 38-41, 52, 55-60, 65, 71, 72, 76, 77, 80, 85, 98, 99, 120, 121, 127, 134, 177, 194, 197 and 199-202 are not compounds of the invention).
- the compounds described herein were evaluated for their ability to interact at the human motilin receptor utilizing a competitive radioligand binding assay as described in Method B1. Further characterization of the interaction can be performed utilizing the functional assays described in Methods B2, B3 and B4. Some of these methods can be conducted, if so desired, in a high throughput manner to permit the simultaneous evaluation of many compounds.
- Other assays have also been described that are suitable for HTS, such as that based upon the stable expression of a synthetic gene for the human motilin receptor.
- Results for the examination of representative compounds of the present invention using Method B1 are presented in Table 3, compounds 1, 2, 5-12, 20, 21, 34, 38-41, 52, 55-60, 65, 71, 72, 76, 77, 80, 85, 98, 99, 127, 134, 177, 194, 197 and 199-202 not being compounds of the invention.
- the assay results of two additional compounds using this Method are shown below. As can be observed, this demonstrates the activity of a representative bicyclic compound of Formula IV, which resulted from incorporation of D-proline as the second recognition building block.
- the compounds are preferably tested in the Aequorin assay as described in Method B2, although the procedure of Method B3 is also applicable.
- the representative compounds examined act as antagonists at the motilin receptor and are devoid of agonist activity at the concentrations studied.
- A 0. 001-0.10 ⁇ M
- B 0.10-1.0 ⁇ M.
- Method B2 measures the actual signaling event, which makes It more relevant to the effect that is desired, whereas the assay of Method B3 simply measures GTP turnover.
- a common and scientifically-accepted ex vivo assay for the measurement of agonist or antagonist activity at the motilin receptor is the contraction of rabbit duodenum or other gastrointestinal smooth muscle tissue.
- A2-A4 Agonists are defined as compounds that induce >50% contraction relative to the motilin peptide, whereas antagonists are defined as compounds that cause >50% inhibition of the response to motilln.
- Gastric motility Is generally measured In the clinical setting as the time required for gastric emptying and subsequent transit time through the Gl tract.
- Gastric emptying scans are well known to those skilled In the art an, briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solid and liquids can be measured independently.
- test food or liquid is radiolabeled with an isotope ( 88m Tc) and after ingestion or administration, transit time through the Gl tract and gastric emptying are measured by visualization using gamma cameras. These studies are performed before and after the administration of the therapeutic agent to quantify the efficacy of the compound.
- Example Method B1 Competitive Radioligand Binding Assay (Motilin Receptor)
- the reaction is initiated by addition of 10 ⁇ l of [ 126 I]-motilin (final conc. 0.04 - 0.06 nM) to each well. Plates are sealed with TopSeal-A, vortexed gently and incubated at room temperature for 2 hours.
- the reaction is arrested by filtering samples through pre-soaked (0.3% polyethyleneimine, 2 h) Multiscreen Harvest plates using a Tomtec Harvester, washed 9 times with 500 ⁇ L of cold 50 mM Tris-HCl (pH 7.4), and than plates are air-dried in a fumehood for 30 minutes. A bottom seal Is applied to the plates prior to the addition of 25 ⁇ L of MicroScint-0 to each well. Plates are than sealed with TopSeal-A and counted for 30 sec per well on a TopCount Microplate Scintillation and Luminescence Counter (PerkinElmer) where results are expressed as counts per minute (cpm).
- Example Method B2 Aequorin Functional Assay (Motilin Receptor)
- Compounds were provided as dry films at a quantity of approximately 1.2 ⁇ mol, in pre-formatted 96-well plates. Compounds were dissolved in 100% DMSO at a concentration of 10 mM and stored at -20°C until further use. Daughter plates were prepared at a concentration of 500 ⁇ M In 30% DMSO with 0.1% BSA and stored at -20°C until testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration In the assay was 0.6%.
- Cells are collected from culture plates with Ca 2+ and Mg 2+ -free phosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelleted for 2 minutes at 1000 x g, resuspended in assay buffer (see above) at a density of 5 x 10 6 cells/mL and incubated overnight in the presence of 5 ⁇ M coelenterazine. After loading, cells were diluted with assay buffer to a concentration of 5'x10 5 cells/mL.
- PBS Ca 2+ and Mg 2+ -free phosphate buffered saline
- assay buffer see above
- test compound or motilin reference agonist
- 50 ⁇ l of the cell suspension was mixed with 50 ⁇ l of the appropriate concentration of test compound or motilin (reference agonist) in 96-well plates (duplicate samples).
- the emission of light resulting from receptor activation was recorded using the Functional Drug Screening System 6000 'FDSS 6000' (Hamamatsu Photonics K.K., Japan).
- an approximate EC80 concentration of motilin i.e. 0.5 nM; 100 ⁇ L was injected onto the cell suspension containing the test compounds (duplicate samples) 15-30 minutes after the end of agonist testing and the consequent emission of light resulting from receptor activation was measured as described in the paragraph above.
- E the measured RLU value at a given agonist concentration (C)
- EC 50 is the concentration producing 50% stimulation
- n is the slope index.
- results for agonist testing results for each concentration of test compound were expressed as percent activation relative to the signal Induced by motilin at a concentration equal to the EC 80 (i.e. 0.5 nM). For antagonist testing, results for each concentration of test compound were expressed as percent inhibition relative to the signal Induced by motilin at a concentration equal to the
- CHO membranes were immobilized into 96-well FlashPlate microplates.
- Test compound, GTP ⁇ S, motilin and [ 35 S]-GTP ⁇ S were combined In each well according to the Assay Volumes described above.
- the reaction is heated by addition of 100 mL of [ 35 S]-GTP ⁇ S to each well.
- Each plate Is sealed (TopSeal-A) and incubated in the dark at room temperature for 150 min. Then, plates are counted for 30 seconds per well on the TopCount NXT.
- Top and Bottom correspond to the top and bottom values of the dose-response curve calculated by GraphPad Prism).
- Duodenal segments were vertically suspended in organ chambers of 10 mL filled with Krebs buffer and connected to an isotonic force transducer, with a preload of 1 g. After a stabilization period, the muscle strips were challenged with 10 -4 M acetylcholine and washed. This was repeated until a stable maximal contraption was obtained (2-3 times), with an Interval of at least 20 minutes.
- test compounds were added to the bath. After 15 min Incubation, a dose response to motilin was recorded by adding logarithmically increasing concentrations of motilin to the bath (final concentration 10 -8 to 10 -6 M). A blank experiment (no test compound present) was also performed. At the end of the dose response curve, a supramaximal dose of acetylcholine (10 -4 M) was given and this response was used as a reference (100% contraction).
- Compound 227, X is Z 1 , Z 2 and Z 3 are NH except for compounds 30, 173 and 174 where Z 1 is O and compound 111 where Z 2 is O.
- R 2 , R 4 and R 5 are hydrogen except for compound 85 where it is: m, n, and p are zero.
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Abstract
Description
- The present invention relates to novel conformationally-defined macrocyclic compounds, pharmaceutical compositions comprising same and intermediates used in their manufacture. More particularly, the invention relates to macrocyclic compounds that have been demonstrated to selectively antagonize the activity of the motilin receptor. The invention further relates to macrocyclic compounds useful as therapeutics for a range of gastrointestinal disorders, in particular those in which malfunction of gastric motility or increased motilin secretion is observed, such as hypermotilinemia, irritable bowel syndrome and dyspepsia.
- A number of peptide hormones are involved in the control of the different functions in the gastrointestinal (GI) tract, including absorption, secretion, blood flow and motility (Mulvihill, et al. in Basic and Clinical Endocrinology, 4th edition, Greenspan, F.S.; Baxter, J.D., eds., Appleton & Lange: Norwalk, CT, 1994, pp 551-570). Since interactions between the brain and GI system are critical to the proper modulation of these functions, these peptides can be produced locally in the GI tract or distally in the CNS.
- One of these peptide hormones, motilin, a linear 22-amino acid peptide, plays a critical regulatory role in the CI physiological system though governing of fasting gastrointestinal motor activity. As such, the peptide is periodically released from the duodenal mucosa during fasting In mammals, including humans. More precisely, motilin exerts a powerful effect on gastric mobility through the contraction of gastrointestinal smooth muscle to stimulate gastric emptying, decrease Intestinal transit time and initiate phase III of the migrating motor complex In the small bowel (Itoh, Z., Ed., Motilin, Academic Press: San Diego, CA, 1990, ASIN: 0123757304; Nelson, D.K. Dig. Dis. Sci. 1996, 41, 2006-2015; Peeters, T.L.; Vantrappen, G.; Janssens, J. Gastroenterology 1980, 79, 716-719).
- Motilin exerts these effects through receptors located predominantly on the human antrum and proximal duodenum, although its receptors are found in other regions of the GI tract as well (Peeters, T.L.; Bormans, V.; Vantrappen, G. Regul. Pept 1989, 23, 71-182). Therefore, motilin hormone is involved In mobility of both the upper and lower parts of the GI system (williams et al. Am. J. Physiol.1992, 262, G50-G55). In addition, motilin and its receptors have been found In the CNS and periphery, suggesting a physiological role in the nervous system that has not yet been definitively elucidated (Depoortere, I.; Peeters, T.L. Am. J. Physiol. 1997, 272, G994-999 and O'Donohue, T.L et al. Peptides 1981, 2, 487.477). For example, motilin receptors In the brain have been suggested to play a regulatory role in a number of CNS functions, including feeding and drinking behavior, micturition reflex, central and brain stem neuronal modulation and pituitary hormone secretion (Itoh, Z. Motilln and Clinical Applications. Peptides 1997, 18, 593-808; Asakawa, A.; Inui, A.; Momose, K.; et al., M. Peptides 1998, 19, 987-990 and Rosenfeld, D.J.; Garthwaite, T.L. Physiol. Behav. 1987, 39, 753-T56). Physiological studies have provided confirmatory evidence that motilin can indeed have an effect on feeding behavior (Rosenfeld, D.J.; Garthwaite, T.L, Phys. Behav. 1987, 39, 735-736).
- The recent identification and cloning of the human motilin receptor (
WO 99/64436 - Due to the critical and direct involvement of motilin in control of gastric motility, agents that either diminish (hypomotility) or enhance (hypermotility) the activity at the motilin receptor, are a particularly attractive area for further investigation in the search for new effective pharmaceuticals towards these indications.
- Peptidlc agonists of the motilin receptor, which have clinical application for the treatment of hypomotility disorders, have been reported (
U.S. 5,695,952 ;5,721,353 ;6,018,037 ;6,380,158 ;6,420,521 ,U.S. Appl. 2001/0041791 ,WO 98/42840 WO 01/00830 WO 02/059141 U.S. 4,920,102 ;5,008,249 ;5,175,150 ;5,418,224 ;5,470,961 ;5,523,401 ,5,554,605 ;5,658,888 ;5,854,407 ;5,912,235 ;6,100,239 ;6,165,985 ;6.403,775 ). - Antagonists of the motilin receptor are potentially extremely useful as therapeutic treatments for diseases associated with hypermotility and hypermotilinemia, including irritable bowel syndrome, dyspepsia, gastroesophogeal reflux disorders, Crohn's disease, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, diabetes mellitus, obesity, malabsorption syndrome, carcinoid syndrome, diarrhea, atrophic colitis or gastritis, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, gastric stasis and eating disorders leading to obesity.
- A variety of peptidic compounds have been described as antagonists of the motilin receptor (Depoortere, I.; Macielag, M.J.; Galdes, A.; Peeters, T. L. Eur. J. Pharmacol. 1995, 286, 241-247;
US 5,470,830 ;6,255,285 ;6,586,630 ;6,720,433 ;U.S. 2003/0176643 ;WO 02/64623 - Cyclization of peptidic derivatives is a method employed to improve the properties of a linear peptide both with respect to metabolic stability and conformational freedom. Cyclic molecules tend to be more resistant to metabolic enzymes. Such cyclic tetrapeptide motilin antagonists have been reported (Haramura, M. et al J. Med. Chem. 2002, 45, 670-675,
U.S. 2003/0191053 ;WO 02/16404 - Other motilin antagonists, which are non-peptidic and non-cyclic in nature have also been reported (
U.S. 5,972,939 ;6,384,031 ;6,392,040 ;6,423,714 ;6,511,980 ;6,624,165 ;6,667,309 ;U.S. 2002/0111484 ;2001/041701 ;2002/0103238 ;2001/0056106 ,2002/0013352 ;2003/0203906 and2002/0002192 ) - In
WO 01/25257 - The macrocyclic motilin antagonists of the present invention comprise elements of both peptidic and non-peptidic structures in a combination which has not been pursued for this application previously.
- Indeed, the structural features of antagonists of the present invention are different. In particular, within the known motilin antagonists which are cyclic peptides, it was found that such derivatives containing D-amino acids were devoid of activity. In contrast, for the tripeptidomimetic compounds of the present invention, the D-stereochemistry is required for two of the three building elements.
- The motilin antagonists of the present invention are also distinct from the prior art in that they comprise a tether element to fulfill the dual role of controlling conformations and providing additional sites for interaction either through hydrophobic interactions, hydrogen bonding or dipole-dipole interactions.
- In a first aspect, the present invention provides a conformationally-defined tripepeptidomimetic macrocyclic compound as defined in claim 1.
-
- Z1, Z2 and Z3 are independently selected from the group consisting of O, N and NR10 , wherein R10 is selected from the group consisting of hydrogen, lower alkyl, and substituted lower alkyl;
- R1 is independently selected from the group consisting of lower alkyl substituted with aryl, lower alkyl substituted with substituted aryl, lower alkyl substituted with heteroaryl and lower alkyl substituted with substituted heteroaryl;
- R2 is hydrogen;
- R3 is independently selected from the group consisting of alkyl and cycloalkyl with the proviso that when Z1 is N, R3 can form a four, five, six or seven-membered heterocyclic ring together with Z1;
- R4 is hydrogen;
- R5 and R6 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl and substituted heteroaryl, with the proviso that at least one of R5 and R6 is hydrogen;
- X Is selected from the group consisting of O, NR8, and N(R9)2 +;
- wherein R8 is selected from the group consisting of hydrogen, lower alkyl, substituted lower alkyl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, sulfonyl, sulfonamide and amidino; and
- R9 is selected from the group consisting of hydrogen, lower alkyl, and substituted lower alkyl;
- m, n1 and p are independently selected from 0,1 or 2; and
- T is a bivalent radical of formula II:
-U-(CH2)d-W-Y-Z-(CH2)8- (II)
- wherein d and e are independently selected from 0, 1, 2, 3, 4 or 5;
- wherein U is bonded to X of formula (I) and is -CH2- or-C(=O)- ;
- wherein Y and Z are each optionally present;
- W, Y and Z are independently selected from the group consisting of: -O-, -NR28-,S-, -SO-, SO2-, -C(=O)-, -C(=O)O-, -O-C(=O)-,-C(=O)-NH-, -NH-C(=O)-, -SO2-NH-, -NH-SO2-, -CR29R30-, -CH=CH- with a configuration Z or E, and -C≡C-, or from a ring structure Independently selected from the group:
- G1 and G2 each Independently represent a covalent bond or a bivalent radical selected from the group consisting of -O-, -NR41-, -S-, -SO-, -SO2-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -C(=O)NH-, -NH-C(=O)-, -SO2-NH-, -NH-SO2-, -CR42R43-, -CH=CH- with a configuration Z or E, and -C≡C-; with the proviso that G1 Is bonded closer to U than G2;
- K1, K2, K3, K4, K6, K15 and K16 are independently selected from the group consisting of O, NR44 and S;
- f is selected from 1, 2, 3, 4, 5 or 6;
- R31, R32, R38, R39, R48 and R49 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido; and
- R33, R34, R35, R36, R37, R47, R50 and R51 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido.
- In a second aspect, the invention provides a process as defined in claim 5.
- In a third aspect, the invention provides a compound as defined in claim 7.
- In his fourth aspect, the invention provides a method as defined in claim 11.
- Preferably in formula (I), as depicted hereinabove, R1 is selected from the group consisting of -(CH2)qR11, and -CHR12R13
wherein q is 0, 1, 2 or 3; and
R11 and R12 are independently selected from a ring structure from the following group: - A1, A2, A3, A4 and A5 are each optionally present and are independently selected from the group consisting of halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido;
- B1, B2, B3, and B4 are independently selected from NR14, S or 0, wherein R14 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, sulfonyl and sulfonamido;
- R13 is as defined for as R11 and R12 or is selected from the group comprising lower alkyl, substituted lower alkyl, hydroxy, alkoxy, aryloxy, amino, carboxy, carboxyalkyl, carboxyaryl, and amido.
-
- Also preferably, R3 in formula (I), is selected from the group consisting of: -(CH2)aCH3, -CH(CH3)(CH2)tCH3, -CH(OR15)CH3, -CH2SCH3 -CH2CH2SCH3, -CH2S(=O)CH3, -CH2CH2S(=O)CH3, -CH2S(=O)2CH3, -CH2CH2S(=O)2CH3, -(CH2)uCH(CH3)2, -C(CH3)3, and -(CH2)y-R21, wherein:
- s and u are independently selected from 0, 1, 2, 3, 4 or 5;
- t Is Independently selected from 1, 2, 3 or 4;
- y is selected from 0, 1, 2, 3 or 4;
- R15 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, formyl and acyl;
- R21 is selected from a ring structure selected from the following group:
- wherein any carbon atom In said ring structure can be replaced by a nitrogen atom, with the proviso that if said ring structure is a monocyclic ring structure, it does not comprise more than four nitrogen atoms and if said ring structure is a bicyclic ring structure, it does not comprise more than six nitrogen atoms;
- z is selected from 1, 2, 3, 4 or 5;
- E1, E2 and E3 are each optionally present and are independently selected from the group consisting of halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido; and
- J is optionally present and is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido.
-
- wherein L1, is O, NH or NMe; L2 is CH or N; L3 is CH or N; L4 is O or CH2; L5 is CH or N L6 is CR52R53 or O; R46 is H or CH3;
- R52, R53, R54, R55, R56 and R57 are independently selected from hydrogen, lower alkyl, substituted lower alkyl, hydroxy, alkoxy, aryloxy, amino, and oxo; or R52 together with R53 or R54 together with R55 or R56 together with R57 can independently form a three to seven- membered cyclic ring comprising carbon, oxygen, sulfur and /or nitrogen atoms;
- (X) is the site of a covalent bond to X in formula (I); and
- (Z3) is the site of a covalent bond to Z3 in formula (I).
- In a particularly preferred embodiment, compounds of formula (I), wherein m, n and p are 0, X, Z1, Z2 and Z3 are NH and R2, R4 and R5 are hydrogen, are represented by formula (III):
n2 is selected from 0, 1, 2 or 3
R7 is optionally present and is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido. - It is to be understood, that in the context of the present invention, the terms amino, guanidine, ureido and amidino encompass substituted derivatives thereof as well.
- Also described herein is a method of treating a disorder associated with hypermotility or hypermotilinemia in humans and other mammals comprising administering a therapeutically effective amount of a compound of formula (I).
-
- PG and PG' indicate a standard amine protecting group compatible with the synthetic protocol, such as Boc, Ddz, Fmoc, or Alloc
- Also described herein is a method of treating Irritable bowel syndrome, dyspepsia, Crohn's disease, gastroesophogeal reflux disorders, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric, stenosis, carcinoid syndrome, matabsorption syndrome, diarrhea, diabetes mellitus, obesity, postgastroenterectomy syndrome, atrophic colitis or gastritis, gastric stasis, gastrointestinal dumping syndrome, celiac disease and eating disorders leading to obesity in humans and other mammals comprising administering a therapeutically effective amount of a compound of formula (I).
- Reagents and solvents were of reagent quality or better and were used as obtained from various commercial suppliers unless otherwise noted. DMF, DCM and THF used are of DriSolv® (EM Science, E. Merck) or synthesis grade quality except for (i) deprotection, (ii) resin capping reactions and (iii) washing. NMP used for the amino add (AA) coupling reactions is of analytical grade. DMF was adequately degassed by placing under vacuum for a minimum of 30 min prior to use. Tyr(3tBu) was synthesized following the method reported in
JP2000 44595 -
- To a solution of the amino acid or amino acid derivative (0.1 mol, 1.0 eq) In 0.25 N sodium hydroxide (0.08 mol, 0.8 eq) with an initial pH of approximately 9.5 (pH meter) at rt, solid Bts-Cl (0.11 mol, 1.1 eq) was added in one portion. The resulting suspension was stirred vigorously for 2-3 d. The pH of the reaction should be adjusted with 5.0 N sodium hydroxide as required to remain within the range 9.5-10.0 during this time. Typically, the pH has to be adjusted every 20-30 min during the first 5 h. Once the pH stops dropping, it is an indication that the reaction is almost complete. This can be confirmed by TLC (EtOAc:MeOH, 95:5). Upon completion, the reaction mixture was washed with Et2O. Washing is continued until the absence of non-polar impurities In the aqueous layer Is confirmed by TLC (typically 3 x 100 mL). The aqueous solution was then cooled to 0°C, acidified to pH 2.0 with 1 N HCl until no additional cloudiness forms, and extracted with EtOAc (3 x 100 mL). Alternatively, a mixture of DCM and EtOAc may be used as the extraction solvent, depending on the solubility of the product obtained from different amino adds or derivatives. Note that DCM cannot be used solely as solvent because of the emulsion formed during extraction. The combined organic phases were washed with brine (2 x 150 mL), dried over MgSO4, filtered and evaporated under reduced pressure. DCM (1x) and hexanes (2x) were evaporated from the residue in order to ensure complete removal of the EtOAc and give the desired compound as a solid in 55-98% yield.
- The following are modifications that have proven useful for certain amino acids:
- Gly, Ala, D-Ala, β-Ala and GABA: Use 1.5 eq of amino acid per eq of Bts-Cl, in order to prevent dibetsylation.
- Met: Carry out the reaction under N2 to prevent oxidation,
- Gln and Asn: Due to the solubility of Bts-Gln and Bts-Asn, the work-up required is modified from the standard procedure: Upon completion of the reaction, the reaction mixture was washed with diethyl ether. Washing is continued until the absence of non-polar Impurities in the aqueous layer is confirmed by TLC (typically 3 x 100 mL). The aqueous phase was then cooled to 0°C and acidified to pH 2.0 with 6 N HCl. 6 N HCl was employed to minimize the volume of the solution due to the water solubility of Bts-Gln and Bts-Asn. (They are, in contrast, difficult to dissolve in DCM, EtOAc or chloroform.) The solution was maintained at 0°C for 10 min and the product was collected by filtration as a white precipitate. The solid was washed with cold water (1x), cold brine (2x) and water (1x, 25°C). The pH of this wash was taken, If It is not approximately 4, the solid was washed again with water. Finally, the solid was washed with cold EtOAc, then with cold Et2O (2x), and finally dried under vacuum (oil pump) (83-85% yield).
-
- The compounds of Formula I can be synthesized using traditional solution synthesis techniques or solid phase chemistry methods. In either, the construction involves four phases: first, synthesis of the building blocks, including one to four moieties, comprising recognition elements for the biological target receptor, plus one tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations. The precursors from the assembly are then cyclized in the third stage to provide the macrocyclic structures. Finally, a post-cyclization processing stage involving removal of protecting groups and optional purification then provides the desired final compounds (Scheme 1). This method has been previously disclosed in
WO 01/25257 U.S. Pat. Appl. Ser. No. 09/679,331 . - The important tether component required for compounds of the invention are synthesized as described in
WO01/25257 U.S. Provisional Pat. Appl. Ser. No. 60/491,248 or herein. Tethers T8, T10, T12, T28, T34, T41 and T42 are not compounds of the invention. -
- Step T8-1: Chlorotrimethylsilane (116 mL, 0.91 mol, 1.5 eq) was added to a suspension of 2-hydroxycinnamic acid (100 g, 0.61 mol, 1.0 eq) in MeOH (500 mL, HPLC grade) over 30 min at 0° C. The resulting mixture was stirred at rt O/N. The reaction was monitored by TLC (EtOAc/MeOH: 98/2). Heating the reaction mixture in a hot water can accelerate the process if necessary. After the reaction was completed, the reaction mixture was evaporated under reduced pressure to afford methyl 2-hydroxycinnamate as a white solid (108.5 g) in quantitative yield. The identity of this intermediate compound is confirmed by NMR. This reaction can be carried out on larger (kg) scale with similar results
- Step T8-2: 3,4-Dihydro-2H-pyran (DHP, 140 mL, 1.54 mol, 2.52 eq) was added dropwise to 2-bromoethanol (108 mL, 1.51 mol, 2.5 eq) In a 2 L three-neck flask with mechanical stirring at 0° C over 2 h. The resulting mixture was stirred for additional 1 h at rt. Methyl 2-hydroxycinnamate from Step T8-1 (108 g, 0.61 mol, 1.0 eq), potassium carbonate (92.2 g, 0.67 mol,1.1 eq). potassium iodide (20 g, 0.12 mol, 0.2 eq) and DMF (300 mL, spectrometric grade) were added to the above flask. The reaction mixture was stirred at 70° C (external temperature) for 24 h. The reaction was monitored by TLC (DCM/Et2O: 95/5). The reaction was allowed to coolto rt and Et2O (450 mL) was added. The inorganic salts were removed by filtration and washed with Et2O (3 x 50 mL). The filtrate was diluted with hexanes (400 mL) and washed with water (3 x 500 mL), dried over MgSO4 filtered and the filtrate evaporated under reduced pressure. The crude ester (desired product and excess Br-C2H4-OTHP) was used for the subsequent reduction without further purification.
- StepT8-3: DIBAL (1.525 L, 1.525 mol, 2.5 eq, 1.0 M in DCM) was added slowly to a solution of the above crude ester from Step T8-2 (0.61 mol based on the theoretical yield) In anhydrous DCM (610 mL) at -35° C with mechanical stirring over 1.5 h. The resulting mixture was stirred for 1.5 h at -35° C, then 1.5 h at 0° C. The reaction was monitored by TLC (hextEtOAc: 50/50). When complete, Na2SO4·10 H2O (100 g, 0.5 eq) was slowly added; hydrogen evolution was observed, when it subsided water was added (100 mL). The mixture was warmed to rt and stirred for 10 min, then warmed to 40° C with hot water and stirred under reflux for 20 min. The mixture was cooled to rt, diluted with DCM (600 mL), and the upper solution decanted into a filter. The solid that remained in the flask was washed with dichloromethane (5 x 500 mL) with mechanical stirring and filtered. The filtrate from each wash was checked by TLC, and additional washes performed if necessary to recover additional product. In an alternative work-up procedure, after dilution with DCM (600 mL), the mixture was filtered. The resulting solid was then continuously extracted with 0.5% TEA in dichloromethane using a Soxhlet extractor. Higher yield was typically obtained by this alternative procedure, although it does require more time. The filtrate was concentrated under reduced pressure and the residue purified by dry pack (EtoAc/hex/Et3N: 20/80/0.5) to give the product alcohol as a yellowish oil (yield: 90%). The identity and purity were confirmed by NMR.
- Step TB-4: To a mixture of the allylic alcohol from Step T8-3 (28 g, 0.100 mol, 1.0 eq) and collidine (0.110 mol, 1.1 eq) In 200 mL of anhydrous DMF under N2 was added anhydrous LICl (4.26 g, 0.100 mol, 1.0 eq.) dissolved in 100 mL of anhydrous DMF. The mixture was then cooled to 0°C, and MsCl (12.67 g, 0.110 mol, 1.1 eq)., freshly distilled over P2O5), was added dropwise. The reaction was allowed to warm to rt and monitored by TLC (3:7 EtOAc/hex). When the reaction was complete, NaN3 (32.7 g, 0.500 mol, 5.0 eq.) was added. The reaction mixture was stirred at rt O/N with progress followed by NMR. When the reaction was complete, the mixture is poured Into an ice-cooled water bath, and extracted with diethyl ether (3x). The combined organic phases were then washed sequentially with citrate buffer (2x), saturated sodium bicarbonate (2x), and finally with brine(1x). The organic layer was dried with MgSO4, filtered and the filtrate concentrated under reduced pressure. The allylic azide was obtained in 90% combined yield, and was of sufficient quality to use as such for the following step.
- Step T8-5: PPh3 (25.9 g, 0.099 mol,1.5 eq) was added at 0° C to a solution of the allylic azide from Step T8-4 (20.0 g, 0.066 mol, 1.0 eq.) in 100 mL of THF. The solution was stirred for 30 min at 0° C and 20 h at rt. Water (12 mL) was then added and the resulting solution was heated at 60° C for 4 h. The solution was cooled to rt, 2N HCl (15 mL) added and the mixture stirred for 90 min at 50°C. The separated organic phase was extracted with 0.05 N HCl (2 x 100 mL). The combined aqueous phase was washed with Et2O (5 x 150 mL) and toluene (4 x 150 mL) (more extraction could be necessary, follow by TLC), which were combined and back-extracted with 0.05 N HCl (1 x 100 mL). This acidic aqueous phase from back-extraction was combined with the main aqueous phase and washed with ether (5 x 150 mL) again. The pH of the aqueous phase was then adjusted to 8-9 by the addition of sodium hydroxide (5 N). Care must be exercised to not adjust the pH above 9 due to the reaction conditions required by the next step. The aqueous phase was concentrated under reduced pressure (aspirator, then oil pump) or lyophilized to dryness. Toluene (2x) was added to the residue and then also evaporated under reduced pressure to remove traces of water. The crude product (desired amino alcohol along with inorgnic salt) was used for the next reaction without further purification.
- Step T8-6: A mixture of the crude amino alcohol from Step T8-5 (0.5 mol based on the theoretical yield), Ddz-OPh (174 g, 0.55 mol, 1.1 eq) and Et3N (70 mL, 0.5 mol, 1.0 eq) in DMF (180 mL) was stirred for 24 h at 50° C. Additional DMF is added if required to solubilize all materials. The reaction was monitored by TLC (hex/EtOAc: 50/50, ninhydrin detection). After the reaction was complete, the reaction mixture was diluted with Et2O (1.5 L) and water (300 mL). The separated aqueous phase was extracted with Et2O (2 x 150 mL). The combined organic phase was washed with water (3 x 500 mL) and brine (1 x 500 mL), dried over MgSO4, filtered and the filtrate concentrated under reduced pressure. The layers were monitored by TLC to ensure no product was lost into the aqueous layer. If so indicated, perform one or more additional extractions with Et2O of the aqueous phase to recover this material, The crude product was purified by dry pack (recommended column conditions: EtOAc/hex/Et3N: 35/65/0.5 to 65/35/0.5) to give the tether Ddz-T8 as a pale yellow syrup (yield: ∼40%). The identity and purity of the product was confirmed by NMR.
1H NMR (DMSO-d6): 1.6 ppm (s, 6H, 2 x CH3), 3.6-3.8 ppm (wide s, 10 H, 2 x OCH3, 2 x OCH2), 3.95 ppm (triplet, 2H, CH2N), 6-6.2 ppm (m, 2H, 2 x CH), 6.2-6.5 ppm (m, 3H, 3 x CH, aromatic), 6.6-7.6 ppm (m, 5H, aromatic). -
- The yield of Ddz-T9 from T9-0 on a 65 g scale was 60.9 g (91%)
1H NMR (CDCl3): □ 7.19-7.01, (m, 2H), 6.92-9.83 (m, 2H), 6.53 (bs, 2H), 6.34 (t, 1H), 5.17 (bt, 1H), 4.08 (m, 2H), 3.98 (m, 2H), 3.79 (s, 6H), 3.01 (bq, 2H), 2.66 (t, 3H), 9.28 (bs, 8H);
13C NMR (CDCl3) □ 160.9, 156.8, 155.6, 149.6, 130.4, 127.5, 121.2, 111.7, 103.2, 98.4, 80., 69.7, 61.6, 55.5, 40.3, 30.5, 29.3, 27.4 - Tether T9 can also be synthesized from T8 by reduction as in step T9-3 or with other appropriate hydrogenation catalysts known to those In the art.
-
- In a dried three-neck flask, a solution of propargylamine (53.7 g, 0.975 mol, 1.5 eq) in degassed DMF (Drisolv, 388 mL) was treated with Ddz-N3 (170.9 g, 0.65 mol, 1.0 eq), tetromethylguanidine (TMG, 81.4 mL, 0.65 mol, 1.0 eq) and DIPEA (113.1 mL, 0.65 mol, 1.0 eq) and stirred at 50°C O/N. The reaction was monitored by TLC (conditions:25/75 EtOAc/hex. Rf 0.25; detection: UV, ninhydrin). Upon completion, DMF was evaporated under reduced pressure until dryness and the residue dissolved in Et2O (1 L). The organic solution was washed sequentially with citrate buffer (pH 4.5, 3x), saturated aqueous sodium bicarbonate (2x), and brine (2x), then dried with MgSO4, filtered and the filtrate evaporated under reduced pressure. A pale orange solid was obtained. This solid was triturated with 1% EtOAc in hex, then collected by filtration and dried under vacuum (oil pump) to provide the desired product (153.4 g, 85.2%).
-
- Two alternative routes to this tether have been developed. The first synthetic approach proceeded starting from the commercially available monobenzoate of resorcinol (T10-0). Mitsunobu reaction under standard conditions with the protected amino alcohol from Example 9, followed by saponification of the benzoate provided T10-1 in good yield after recrystallization. Alkylation of the phenol with 2-bromosthanol using the optimized conditions shown permitted the desired product Ddz-T10 to be obtained after dry pack purification in 42% yield.
TLC (EtOAc/Hexanes 1:1, detection: UV, ninhydrin; Rf = 0.17)
1H NMR (CDCl3) δ 7.18, t, 1H,J = 8.2Hz; 6.51, m, 5H; 6.34, t, 1H, J = 2.2Hz; 5.19, s, 1H; 4.05, t, 2H, d = 5.0Hz; 3.94, m, 4H; 3.75, s, 6H; 3.49, d, 2H J = 5.2Hz;1.73, s, 6H.
13C NMR (CDCl3) δ 160.858; δ 160.152; 160.005; 155.410; 149.305; 130.279; 107.438; 107.310; 103.163; 101.877; 98.517; 69.488; 67.382; 61.595; 55.427; 40.420; 29.427.
HPLC (standard gradient) tR: 7.25 min
MS: 420 (M+H) -
- From resorcinol, two successive Mitsunobu reactions are conducted with the appropriate two carbon synthons illustrated, themselves derived from 2-aminoethanol and ethylene glycol, respectively, through known protection methodologies. Lastly, deprotection of the silyl ether, also under standard conditions provided Boc-T10.
- Although the yields in the two methods are comparable, the first required less mechanical manipulation and is preferred for larger scales.
-
- TLC (15:85 THF/DCM; detection: UV; Rf: 0.33).
1H NMR (DMSO-d8) δ 8.00, d, 1H; 7.32, d, 1H; 7.15, m, 1H; 6.44, s, 2H; 6.33, s, 1H; 3.99, t, 2H; 3.71, m, 8H; 2.89, m =4, 2H; 2.71, t, 2H; 1.71, m = 5, 2H; 1.61, s, 8H.
13C NMR, solvent DMSO-d6) δ 160.879; 153.275; 151.405; 150.447; 140.773; 122.666; 118.934; 103.347; 98.456; 79.778; 70.449; 60.212; 55.717; 55.599; 29.740; 28.592.
HPLC (standard gradient) tR: 5.4 min
MS: 419 (M+H) -
- In a 3-L flame-dried three-neck flask, a solution of (aminomethyl)phenylthiobenzyl alcohol (12-0, 96 g, 0.39 mol) in degassed DMF (1 L, 0.4 M) was prepared. To this was added DdzN3 (0.95 eq), followed by TMG (0.39 mol, 49 mL). The reaction was stirred for 10 min, then DIPEA (68 mL, 0.39 mol) added. The mixture was heated at 50°C under N2 until TLC indicated no DdzN3 remained (48 h typically). (TLC eluent: EtOAc:Hex 50:50; detection: ninhydrin). Upon completion, to the reaction mixture was added 3 L citrate buffer and the separated aqueous layer extracted with C-t2O (3 x 1500 mL). The combined organic phase was washed sequentially with citrate buffer (2 x 200 mL), water (2 x 200 mL) and brine (2 x 200 mL). The organic layer was dried over MgSO4, filtered and the filtrate evaporated under reduced pressure. A dark orange oil was obtained, which was purified by dry-pack. For this procedure, the oil was first dissolved in EtOAc:Hex:DCM:TEA (20:80:1:0.5, vlvlv/v). At this point, a little extra DCM was sometimes required to ensure complete dissolution. The solution was loaded onto the column, then the column eluted with EtOAc:Hex:DCM:Et3N (20:80:1:0.5) until all the impurities were separated out as Indicated by TLC, paying particular attention to that closest to the desired product The elution was then continued with EtOAc:Hex:Et3N 30:70:0.5 (v/v/v) and finally with EtOAc:hexanes:Et3N (50:50:0.5) to elute the desired product. After removal of the solvent from the fractions containing the product under reduced pressure, the residue was dissolved in the minimum amount of DCM, a three-fold larger volume of hexanes added, then the solvents again evaporated under reduced pressure. This treatment was repeated until an off-white foam was obtained. The latter solidified while drying under vacuum (oil pump). Alternatively, the material yielded a solid after sequential concentration with DCM (1x) and hexanes (2x). Tether Ddz-T12 was obtained as an off-white solid (85-90% yield).
- To a 0.2 M solution of the appropriate tether (1.5 eq) in THF or THF-toluene (1:1) was added the PPh3-DIAD (pre-formed by mixing equivalent amounts of the reagents and isolated by evaporation of solvent, see Example 29-C) adduct (1.0 eq.). The resultant mixture was manually agitated for 10 sec (the solution remained turbid), then added to the resin. Alternatively, the resin was added to the solution. The reaction suspension was agitated O/N (after ~5 min the mixture becomes limpid). The resin was filtered and washed 2x DCM, 1x toluene, 1x EtOH, 1x toluene, 1x (DCM/MeOH), 1x (THF/MeOH), 1x (DCM/MeOH), 1x (THF/MeOH), 2x DCM, then dried in the standard manner.
- To a 0.2 M solution of the appropriate tether (4 eq) in THF or THF-toluene (1:1) was added triphenylphosphine (4 eq). The resultant mixture was manually shaken until a homogenous solution was obtained, then added to the resin. Alternatively, the resin (or MiniKans containing resin) was added to the solution. To this suspension was then added DIAD (3.9 eq) and the reaction agitated O/N. Note: Since the reaction is exothermic, for larger scales, the reaction should be cooled in an Ice bath. In addiction, an appropriate vent must be supplied to allow any pressure build-up to be released. The resin was filtered and washed DCM (2x), toluene (1x), EtOH (1x), toluene (1x), DCM/MeOH (1x), 1x THF/MeOH (1x), DCM/MeOH (1x), THF/MeOH (1x), 2x DCM, then dried in the standard manner.
-
- DIAD (1 eq) was added dropwise to a well-stirred solution of triphenylphosphine (1 eq) in THF (0.4 M) at 0°C under nitrogen. The mixture was then maintained at 0°C with stirring for 30 min. The white solid obtained was collected by filtration (use medium sized fritted filters), washed with cold anhydrous THF until the washes were colorless, and lastly washed once with anhydrous Et2O. The white solid product was then vacuum-dried (oil pump) and stored under nitrogen. (Note: The PPh3-DIAD adduct can be made In larger than immediately required quantity and stored under nitrogen; It is very important to store this reagent under anhydrous conditions.)
- In certain instances, the Mitsunobu process of Example 29 cannot be applied or is not efficient for incorporation of the tether. Hence, reductive amination has been developed as an alternative that can be employed for tether incorporation as Illustrated hereinbelow for one of the preferred tethers. Similar chemistry can be used to Incorporate other tethers of the present invention.
- The Tether (30-2) with the amine protected as its Ddz derivative was efficiently oxidized to the corresponding aldehyde 30-2 using SO3·pyr in DMSO-Et3N-DCM. This aldehyde (0.14 mmol, 56 mg, 1.5 eq based upon loading of resin support) was dissolved in a 1:3 mixture of TMOF-MeOH (DriSolv, 4 mL) at rt. To this was added the resin containing the tripeptide (30-1, as its trifluoroacetic acid salt from the deprotection of the terminal amine), the mixture was agitated briefly to wet the resin, and then borane-pyridine complex (as the commercially available 8 M solution, 23 µL, 2 eq) was introduced to the suspension. The reaction was agitated O/N, then the resin filtered, washed with DCM (2x), THF (1x), DCM/MeOH [3:1] (1x), THF/MeOH [3:1] (1x), DCM (2x) and dried in the standard manner. Care must be taken to ensure that the desired resin bound product 30-3 is not contaminated with the dialkylated material. However, even if the reaction does not proceed to completion or if a small amount of the dialkylation side product is present, the material is of sufficient purity for the macrocyclization reaction.
-
- Henry reaction of 2-hydroxybenzaldehyde 28-0 provided 28-1 in 79% yield. This was followed by reduction first with sodium borohydride, then with catalytic hydrogenation, to give the amine, which was then protected as its Boc derivative, 28-2. Yields of these first two steps were lower on larger scales. Alkylation of 28-2 with the TBDMS ether of 2-bromoethanol, itself synthesized by standard methods, gave 28-3 in 74% yield. Deprotection of the silyl ether under standard conditions, yielded the desired protected tether, Boc-T28. Alternative use of ethylene carbonate for the phenol alkylation to avoid the protection/deprotection steps, gave 73% yield.
-
- TLC (100% EtOAc; detection: UV, CMA; Rf = 0.24).
1H NMR (CDCl3, ppm): 7.74 (1H, dd), 7.35 (1H, d), 6.72 (1H, d), 6.53-6.49 (2H, m), 3.61-3.29 (1H, m), 5.06 (1H, t), 4.25-4.01 (2H, m), 3.91-3.89 (2H, m), 3.73 (3H, s), 2.99 (2H, dd), 2.63 (2H, t), 1.71 (8H, broad),1.53 (9H, s).
13C NMR (CDCl3, ppm): 163.8, 162.2, 181.0,168.7,155.9, 149.4, 130.0,128.1,128.0, 126.8,110.8, 98.1, 80.9,79.3, 69.7, 61.3, 55.5,39.1,29.3,28.5, 26.7. -
- The construction to the (R)-isomer of this tether (T33a) was accomplished from 2-iodophenol (33-0) and (S)-methyl lactate (33-A). Mitsunobu reaction of 33-0 and 33-A proceeded with inversion of configuration in excellent yield to give 33-1. Reduction of the ester to the corresponding alcohol (33-2) also occurred In high yield and was followed by Sonagashira reaction with Ddz-propargylamine. The alkyne In the resulting coupling product, 33-3, was reduced with catalytic hydrogenation. Workup with scavenger resin provided the desired product, Ddz-T33a.
1H NMR (CDCl3) δ (ppm) 7.18-7.11 (m, 2H), 6.90 (m, 2H), 6.52 (m, 2H), 6.33(m, 1H), 6.09 (bt, 1H), 4.52 (m, 1H), 3.77 (s, 6H), 3.08 (bq, 2H), 2.64 (bt, 2H), 1.75 (m, 8H); 1.27 (bd, 3H), 13C NMR (CDCl3) δ 160.8,155.5,149.5,131.2,130.6,127.4,121.2,113.3, 103.2, 98,4, 80.7, 74.8, 66.5, 55,4, 40.2, 30.6, 29.3, 29.2, 27.4, 16.1
HPLC (standard gradient): tR: 7.93 min -
-
- TLC (100% EtOAc; detection: CMA, Rf = 0.5).
MW Calc. for C24H35N3O7, 477.55; MS Found (M+H)+ 478.
1H NMR (CDCl3) δ1.62 (m, 2H), 1.70 (m, 8H), 2.43 (m, 2H), 2.67 (m, 2H), 3.07 (m, 2H), 3.34 (s. 3H), 3.43 (s, 3H), 3.61 (m, 2H), 3.75 (s, 6H), 5.40 (sb, 1H), 6.31 (s,1 1H), 6.49 (s, 2H)
13C NMR (CDCl3) δ23.25 (CH2), 25.97 CH2), 28.56 (CH3), 39.31 (CH3), 30.09 (CH3), 31.25 (CH2). 32.19 (CH2), 40.16 (CH2). 55.47 (CH3). 61.38 (CH2), 80.65 (Cq). 99.38 (Cq),103.17 (Cq), 111.01(Cq), 149,60 (Cq), 151.33 (Cq), 152.46 (Cq), 160.80 (Cq).
HPLC (standard gradient) tR: 6.68 min. -
- TLC (25/75 EtOAc/Hex; detection: UV, ninhydrin; Rf = 0.03)
1H NMR (CDCl3): δ 7,06-7.00 (bt, 1H), 6.61-6.52 (m, 4H), 6.35 (m, 1H), 5.12 (bt, 1H), 4.03 (m, 2H), 3.95 (m, 2H), 3.77 (s, 6H), 3.11-3.04 (bq, 2H), 2.60 (bt, 2H), 1.75 (m, 8H)
13C NMR (COCl3): δ 163.9, 160.9, 160.0, 157.6, 157.5, 165.6, 149.5, 130.8, 130.6, 125.9, 107.28, 108.9, 103.2, 98,4, 80.8, 77.5, 69.9, 61,3, 60.9, 60.6, 55,4, 40.3, 30.4, 29.3, 26.9,
HPLC (standard gradient): tR = 8.37 min -
- TLC: (25/75 EtOAc/Hex; detection: UV, ninhydrin; Rf = 0.03)
1H NMR (CDCl3) 6 (ppm): 6.84-6.75 (m, 3H), 6.52 (bs, 2H), 6.34 (m, 1H), 5.17 (bt, 1H), 4.01 (m, 2H), 3.93 (m, 2H), 3.77 (s, 6H), 3.10 (bq, 2H), 2.63 (bt, 2H), 1.74 (m, 8H)
13C NMR (CDCl3) δ 160.9, 158.9, 155.8, 155.6, 152.9, 152.9, 149.5, 132.4, 132.3, 117.1, 116.8, 112.7, 112.6, 103.2, 98.4, 80.8, 70.4, 61.6, 55.5, 40.2, 30.3, 29.3, 27.4.
HPLC (standard gradient): tR = 8.29 min -
- TLC (25/75 EtOAc/Hex; detection: UV, ninhydrin; Rf = 0.03)
1H NMR (CDCl3): δ 7.12-7.08 (bd, 2H), 6.76-6.73 (d, 1H), 6.52 (m, 2H), 6.33 (bs, 1 H), 5.15 (bt, 1 H), 4.02 (m, 2H), 3.95 (m, 2H), 3.79 (s, 6H), 3.09 (bq, 2H), 2.61 (bt, 2H), 1.74 (m, 8H). 13C NMR (CDCl3) δ 160.8, 155,6, 155.4, 149.5, 132.4, 130.1, 127.0, 126.0, 112.8,103.2, 98.4, 80.8, 70.0, 61.4, 55.5, 40.3, 30.2, 29.3, 24.5, 27.4
HPLC (standard gradient): tR = 9.60 min -
- 1H NMR (CDCl3): δ 7.20-7.10. (m, 2H), 6.95-6.80 (m, 2H), 6.55 (bs, 2H), 6.35 (s, 1H), 5.18 (bt, 1H), 4.12 (m, 1H), 3.95 (m, 2H), 3.80 (s. 6H), 3.15 (bq, 2H), 2.65 (t, 2H), 1.98 (bs, 2H), 1.65 (bs, 6H), 1.25 (m, 3H).
13C NMR (CDCl3); δ160.8, 156.6, 155.8, 149.8, 130.4, 127.5, 121.3, 111.7, 103.2, 98.4, 80.7, 73.5, 66.6, 55.5, 40.2, 30.5, 29.3, 29.1, 27.3, 19.5.
Chiral T38 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature.
HPLC (standard gradient) tR = 8.46 min - Chiral material can be accessed by starting with the chiral epoxide. For example, the (S)-isomer of T38 was constructed in 89% overall yield from (S)-propylene oxide.
-
- TLC (50% EtOAc, 50% Hex; detection: UV, and CMA; Rf = 0.25)
1H NMR (CDCl3, ppm): 7.11-7.08 (2H, m), 6.86 (1H, t), 6.76 (1H, d), 5.05 (1H, broad), 4.26-3.85 (4H, m), 3.22-3.07 (2H, m), 2.71 (1H, broad), 1.66-1.60 (2H, m), 1.33 (9H. s), 1.17 (3H, d).
13C NMR (CDCl3, ppm): 156.1, 135.0, 127.1, 127.0, 121.4, 111.7, 69.9,61.5, 39,8, 38.4,28.7,20.7. - Chiral T39 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature.
-
- TLC (50% EtOAc, 50% Hex; detection: UV and CMA; Rf = 0.25)
1H NMR (CDCl3, ppm): 7.11-7.08 (2H, m), 6.86 (1H, t), 6.76 (1 H, d), 5.05 (1H, broad), 4.26-3.85 (4H, m), 3.22-3.07 (2H, m), 2.71 (1 H, broad), 1.66-1.60 (2H, m), 1.33 (9H, s), 1.17 (3H, d).
13C NMR (CDCl3, ppm): 156.1, 135.0, 127.1, 127.0, 121.4, 111.7, 69.9, 61.5, 39.8, 38.4, 28.7, 20.7. - Chiral T40 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature.
-
- TLC (100% EtOAc; detection: CMA; Rf= 0.5)
1H NMR (CDCl3) δ.1,23 (s, 3H), 1.49 (s, 3H), 1.69 (s, 3H), 1.74 (s, 3H), 1.90 (m, 2H), 2.35 (m, 1 H), 3.35 (m, 2H), 3.76 (s, 6H), 3.92 (m, 2H), 4.40 (m, 2H), 5.10 (m, 1H), 6.15 (s, 1H), 6.25 (s, 2H).
13C NMR (CDCl3) δ25.52 (CH3), 27.53 (CH3), 28.88 (CH3), 29.61 (CH3), 35.92 (CH2), 42.62 (CH2) , 55.43 (CH3), 60.60 (CH2), 82,38 (CH), 83.33 (CH), 83.68 (CH), 84.96 (CH), 98.26 (CH), 103.23 (CH), 118.3 (Cq), 149.50 (Cq), 156.20 (Cq), 160, 02 (Cq)
HPLC (standard gradient): tR = 6.64 min
MS: M+H found: 439 -
- 1H NMR (300 MHz, CDCl3) δ 6.82-6.98 (m, 2H); 6.80-6.75 (m, 1H); 6.53 (s, 2H); 6.35 (t, 1 H, 2 Hz); 5.23 (b, 1H): 4.08 (m, 1 H); 3.90-3.68 (m, 8H); 3.20-2.97 (m, 2H); 2.95-53 (m, 4H); 2.0-1.63 (m, 10H).
13C NMR (75.5 MHz, CDCl3) δ160.85; 155.56; 152.55; 149.56; 128.13; 127.77; 120.28; 103.22; 98.43; 80.72; 76.80; 65.76; 55.46; 40.23; 30.45; 29.34; 29.22; 27.10; 24.97; 23.94. -
- One or more of the amino acids indicated can be replaced by corresponding hydroxy acids and coupled to the next building block utilizing methods known to those in the art.
- The resin containing the cyclization precursor is combined in an appropriate vessel with pre-washed MP-carbonate resin [Argonaut Technologies, Foster City, CA, commercially supplied MP-carbonate resin was treated with 3x THF (1 L per 400 g) and dried O/N at 30°C in a vacuum oven] (1.4 to 1.6 eq relative to the initial loading of the synthesis resin). A 0.2 M DIPEA solution in THF was then added to the combined resins (1 mL / 60 mg MP-carbonate resin) and the suspension agitated O/N at rt. Subsequently, the resin was filtered and rinsed 2x THF. The combined filtrates are collected together in an appropriate vessel, then the volatile contents evaporated under reduced pressure [in addition to the standard methods, solvent can also be removed in vacuo using centrifugal evaporation (ThermoSavant Discovery, SpeedVac or comparable)] to provide the crude macrocycles.
- Except for the cyclization itself and subsequent work-up, this procedure is Identical to that of Example 47. The resin containing the cyclization precursor was combined in an appropriate vessel with pre-washed MP-carbonate resin [Argonaut Technologies, commercially supplied MP-carbonate resin was treated with THF (3x, 1 L per 400 g) and dried O/N at 30°C in a vacuum oven] (1.4 to 1.6 eq relative to the initial loading of the synthesis resin). To this was added THF (1 mL per 100 mg resin) and silver trifluoroacetate (1 eq relative to the initial loading of the resin). Finally, an amount of DIPEA sufficient to obtain a 0.2 M solution was added. The reaction mixture was agitated at rt O/N. The solution was then filtered and the resins washed 2x THF. The filtrates are collected together in an appropriate vessel, then evaporated under reduced pressure [(the volatile contents could also be removed in vacuo using centrifugal evaporation (ThermoSavant Discovery, SpeedVac or comparable)] to provide the crude macrocycles. For this procedure, silver trifluoroacetate should be stored in a desslcator between uses. In addition, it is recommended to use a new bottle of THF (or a bottle that has been recently opened under N2 or Ar) to minimize formation of sliver oxide.
- Additionally, a ring-closing metathesis (RCM) strategy, as developed by Grubbs et al. can also be used to access some of the macrocyclic compounds of the invention (see for example
US 5,811,515 ; Grubbs, R.H. et al. J. Org. Chem. 2001, 66, 5291-5300; Fürstner, A. Angew. Chem. Int. Ed. 2000, 39, 3012-3043). - To access certain derivatives of compounds of the present invention, additional reactions from those in the general scheme were required. For some, It was advantageous to react the functionality to be derivatized prior to the formation of the macrocyclic ring. The cyclic structure can restrict access of reagents to that functionality. For example, in the synthesis of N-methyl and N-acyl derivatives of macrocycles, where the secondary nitrogen atom of the ring is the site of derivatization, the reaction is preferred to be performed prior to the application of the appropriate cyclization protocol.
- In other cases, for example the derivatization of side chain functionality, the reaction was best performed after formation of the macrocyclic ring. For example, further reaction of amino moieties on side chains examples was typically efficiently done by reaction of the partially protected macrocycle. In this manner, acylation, sulfonylation, alkylation (via reductive amination), guanidine and urea formation were performed via standard methods. Table 1, hereinbelow, shows a representative, but by no means exclusive, summary of the chemical synthesis of several representative compounds of the invention (compounds 1, 2, 5-9, 11, 12, 20, 21, 34, 38-41, 52, 55-60, 65, 71, 72, 76, 77, 80, 85, 98, 99, 120, 121, 127, 134, 177, 194, 197 and 199-202 not being compounds of the invention).
Table 1: Synthesis of Representative Compounds of the Present Invention AA1 AA2 AA3 Tether Tether Attachment Additional Steps 1 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T8 Example 29 none 2 Bts-D-Phe Boc-D-Val Boc-Nva Boc-T8 Example 29 none 3 Bts-D-Phe Boc-D-Val Boc-Nva Boc-T9 Example 29 none 4 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 5 Bts-D-Tyr(tBu) Boc-D-Ala Boc-Nva Ddz-T8 Example 29 none 6 Bts-D-Tyr(tBu) Boc-D-Val Boc-Met Ddz-T8 Example 29 none 7 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nle Ddz-T8 Example 29 none 8 Bts-D-Tyr(tBu) Boc-D-Val Boc-Phe Ddz-T8 Example 29 none 9 Bts-D-Tyr(tBu) Boc-D-Val Boc-Val Ddz-T8 Example 29 none 10 Bts-D-Tyr(tBu) Boc-D-Val Boc-Leu Ddz-T9 Example 29 none 11 Bts-D-2-Nal Boc-D-Val Boc-Nva Boc-T8 Example 29 none 12 Bts-D-Tyr(tBu) Boc-D-Val Boc-Abu Ddz-T8 Example 29 none 13 Bts-D-Phe Boc-D-Val Boc-Leu Boc-T9 Example 29 none 14 Bts-D-2-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 none 15 Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 16 Bts-D-Phe(4Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 17 Bts-D-Trp(Boc) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 18 Bts-D-Tyr(tBu) Boc-D-2-Abu Boc-Nva Ddz-T9 Example 29 none 19 Bts-D-Phe(4F) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 20 Bts-D-Phe Boc-D-Val Boc-Leu Boc-T8 Example 29 none 21 Bts-D-2-Nal Boc-D-Val Boc-Leu Boc-T8 Example 29 none 22 Bts-D-Tyr(OMe) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 23 Bts-D-1-Nal Boc-D-Val Boc-Nva Boc-T9 Example 29 none 24 Bts-D-2-Thi Boc-D-Val Boc-Nva Boc-T9 Example 29 none 26 Bts-D-Phe(2Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 26 Bts-D-Tyr(tBu) Boc-D-Val Boc-Cpa Ddz-T9 Example 29 none 27 Bts-D-4-Thz Boc-D-Val Boc-Nva Boc-T9 Example 29 none 28 Bts-D-3-Pal Boc-D-Val Boc-Nva Boc-T9 Example 29 none 29 Bts-D-Tyr(tBu) Boc-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 30 Bts-D-Tyr(tBu) Hnva(THP) Boc-Nva Ddz-T9 Example 29 none 34 Bts-D-Tyr(tBu) Ddz-D-Tyr(tBu) Boc-Nva Ddz-T8 Example 29 None 38 Bts-D-Tyr(tBu) Boc-D-Val Boc-Ala Ddz-T8 Example 29 none 39 Bts-D-Tyr(tBu) Boc-D-Val Boc-□-Ala Ddz-T8 Example 29 none 40 Bts-D-Tyr(tBu) Boc-D-Val Boc-Gly Ddz-T8 Example 29 none 41 Bts-D-Tyr(tBu) Boc-DPhe Boc-Nva Ddz-T8 Example 29 none 52 Bts-D-Tyr(tBu) Boc-D-Val Boc-Phg Ddz-T8 Example 29 none 55 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Lys(Boc) Ddz-T8 Example 29 none 56 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Orn (Boc) Ddz-T8 Example 29 none 57 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Ser(tBu) Ddz-T8 Example 29 none 58 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Tyr(tBu) Ddz-T8 Example 29 none 59 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Trp(Boc) Ddz-T8 Example 29 none 60 Bts-D-Tyr(tBu) Boc-D-Val Boo-Tyr(OMe) Ddz-T8 Example 29 none 65 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T2 Example 29 none 71 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T10 Example 29 none 72 Bts-D-Tyr(tBu) Boc-D-Val Boc-2-Nal Ddz-T8 Example 29 none 76 Bts-D-Tyr(tBu) Boc-D-2-Nal Boc-Nva Ddz-T8 Example 29 none 77 Bts-D-Tyr(tBu) Boc-D-Nle Boc-Nva Ddz-T8 Example 29 none 80 Bts-D-Tyr(tBu) Boc-D-Val Boc-Ile Ddz-T8 Example 29 none 86 Bts-D-Tyr(tBu) Boc-D-Val Boc-D-Nva Ddz-T8 Example 29 none 87 Bts-D-Bip Boc-D-Val Boc-Nva Boc-T9 Example 29 none 88 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 89 Bts-D-Hfe Boc-D-Val Boc-Nva Boc-T9 Example 29 none 90 Bts-D-Dip Boc-D-Val Boc-Nva Boc-T9 Example 29 none 91 Bta-D-Tyr(tBu) Boc-D-Nva Boc-Nva Ddz-T9 Example 29 none 92 Bts-D-Tyr(tBu) Boc-D-Tle Boc-Nva Ddz-T9 Example 29 none 96 Bts-D-Tyr(tBu) Boc-β-Ala Boc-Nva Ddz-T9 Example 29 none 97 Bts-D-Tyr(tBu) Boc-D-Chg Boc-Nva Ddz-T9 Example 29 none 98 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T18 Example 29 none 99 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T15 Example 29 none 109 Bts-D-Tyr(tBu) Boc-D-Val Ddz-Dab(Boc) Ddz-T9 Example 29 none 110 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T11 Example 29 none 111 Bts-D-Tyr(tBu) Boc-D-Val Hval(THP) Ddz-T9 Example 29 none 112 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ddz-T9 Example 29 none 120 Bts-D-Tyr(tBu) Boc-D-Pro Boc-Nva Ddz-T8 Example 29 none 121 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Ac-T8-NH2 Example 29 none 122 Boc-D-3-Pal Boc-D-Val Boc-Nva Boc-T9 Example 30 none 123 Boc-D-2-Pal Boc-D-Val Boc-Nva Boc-T9 Example 30 none 124 Boc-D-4-Pal Boc-D-Val Boc-Nva Boc-T9 Example 30 none 126 Bts-D-Tyr(tBu) Boc-D-Cpg Boc-Nva Boc-T9 Example 29 none 126 Bts-D-Tyr(tBu) Boc-D-Val Boc-NMeLeu Boc-T9 Example 29 none 127 Boc-D-His(Mts) Boc-D-Val Boc-Nva Boc-T12 Example 30 none 128 Bts-D-Tyr(OM e) Boc-D-Val Boc-Leu Boc-T9 Example 29 none 129 Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 none 130 Bts-D-2-Thi Boc-D-Val Boc-Leu Boc-T9 Example 29 none 131 Bts-D-Phe(3Cl ) Boc-D-Val Boc-Leu Boc-T9 Example 29 none 132 Bts-D-Phe(4Cl ) Boc-D-Val Boc-Leu Boc-T9 Example 29 none 133 Bts-D-Phe(4F) Boc-D-Val Boc-Leu Boc-T9 Example 29 none 134 Bts-D-Phe(3Cl Boc-D-Val Boc-Leu Boc-T2 Example 29 none 135 Bts-D-Tyr(OM e) Boc-D-Val Boc-Leu Boc-T11 Example 29 none 136 Bts-D-1Nal Boc-D-Val Boc-Leu Boc-T11 Example 29 none 137 Bts-D-2-Thi Boc-D-Val Boc-Leu Boc-T11 Example 29 none 138 Bts-D-Phe(3Cl ) Boc-D-Val Boc-Leu Boc-T11 Example 29 none 139 Bts-D-Phe(4Cl) Boc-D-Val Boc-Leu Boc-T11 Example 29 none 140 Bts-D-Phe(4F) Boc-D-Val Boc-Leu Boc-T11 Example 29 none 141 Bts-D-Tyr(OMe) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 142 Bts-D-1-Nal Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 143 Bts-D-2-Thi Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 144 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 145 Bts-D-Phe(4Cl) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 146 Bts-D-Phe(4F) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 147 Bts-D-Tyr(OMe) Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 148 Bts-D-1-Nal Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 149 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 150 Bts-D-Phe(4Cl) Boc-D-Val Boc-Cpa Boc-T11 Example 29 none 151 Bts-D-Phe(4F) Boc-D-Val Boc-Cpa Boo-T11 Example 29 none 152 Bts-D-Tyr(OMe) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 153 Bts-D-1-Nal Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 154 Bts-D-2-Thl Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 155 Bts-D-Phe(3Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 156 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 157 Bts-D-Phe(4F) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 none 158 Bts-D-Phe(3Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T11 Example 29 none 159 Bta-D-Tyr(But) Boc-D-Ile Boc-Nva Boc-T9 Example 29 none 160 Bts-D-Tyr(But) Boc-D-allolle Boc-Nva Boc-T9 Example 29 none 161 Boc-D-Phe(4C H2NHF moc) Boc-D-Val Boc-Nva Boc-T9 Example 30 none 162 Bts-D-Phe(2M e) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 163 Bts-D-Phe(3M e) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 164 Bts-D-Phe(4M e) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 165 Bts-D-Phe(3O Me) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 166 Bts-D-Phe(2O Me) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 167 Bts-D-3-benzothi enyl Boc-D-Val Boc-Nva Boc-T9 Example 29 none 168 Bts-D-3-Thi Boc-D-Val Boc-Nva Boc-T9 Example 29 none 169 Bts-D-□-HomoP he(3Cl) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 170 Bts-D-Phe(3,4 diCl) Boc-D-Val Boo-Nva Boc-T9 Example 29 none 171 Bts-D-Phe(3,4 diF) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 172 Bts-D-Phe(3,4 diOMe) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 173 Bts-D-1Nal Hnva(THP) Boc-Nva Boc-T9 Example 29 none 174 Bts-D-Tyr(OM e) Hnva(THP) Boc-Nva Boc-T9 Example 29 none 175 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Boc-T33b Example 29 none 176 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Boc-T33a Example 29 none 177 Bts-D-Tyr(tBu) Boc-D-Val Boc-Nva Boc-T28 Example 29 none 178 Bts-D-Tyr(OM ) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 179 Bts-D-1-Nal Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 180 Bts-D-2-Thi Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 181 Bts-D-Phe(3Cl) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 182 Bts-D-Phe(4Cl ) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 183 Bts-D-Phe(4F) Ddz-D-Val Ddz-Ser(tBu) Ddz-T9 Example 29 none 184 Bts-D-1-Nal Ddz-D-Val Ddz-Dap(Boc) Ddz-T11 Example 29 none 185 Bts-D-Phe(4Cl ) Ddz-D-Val Ddz-Dap(Boc) Ddz-T11 Example 29 none 186 Ddz-D-Tyr(tBu) Ddz-D-Val His(Mts) Ddz-T9 Example 30 none 187 Bts-D-Phe(3C F3) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 188 Bts-D-Phe(3F) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 189 Bts-D-Phe(4N O2) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 190 Bts-D-3-benzothi enyl Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 191 Bts-D-Phe(3O Me) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 192 Bts-D-Phe(3,4 diCl) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 193 Bts-D-Phe(3,4 diF) Boc-D-Val Boc-Cpa Boc-T9 Example 29 none 194 Bts-D-Tyr(OM e) Boc-D-Val Boc-Nva Boc-T34 Example 29 none 195 Bts-D-Tyr(OMe) Boc-D-Val Boc-Nva Boc-T38 Example 29 none 196 Phe(3Cl) Boc-D-Val Boc-Cpa Ddz-T32(Boc) Example 29 none 197 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T34 Example 29 none 198 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T38 Example 29 none 199 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T41 Example 29 none 200 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T8 Example 29 none 201 Bts-D-1-Nal Boc-D-Val Boc-Nva Boc-T8 Example 29 none 202 Bts-D-Phe(3O Me) Boc-D-Val Boc-Nva Boc-T8 Example 29 none 203 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 acetylation 204 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 guanidinylation 205 Bts-D-Phe(3Cl) Boc-D-Val Boc-NMeLeu Boc-T9 Example 29 none 208 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 mesylation 207 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 TMS-isocyanate followed by dilute acid 208 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 guanidinylation 209 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 acetylation 210 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 reductive amination with acetone 211 Bts-D-Phe(4Cl) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 reductive amination with excess formaldehyde 212 Bts-D-Phe(4C) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 reductive amination with acetone 213 Bts-D-Tyr(3,5dil) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 214 Bts-D-Tyr(OMe) Boc-D-Val Boc-Hse(Bzl) Boc-T9 Example 29 hydrogenoly sis for protecting group removal 215 Bts-D-Tyr(tBu) Ddz-D-Val Ddz-Dap(Boc) Ddz-T9 Example 29 reductive amination with excess formaldehyde Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T40 Example 29 none 217 Bts-D-Phe(3Cl) Boc-D-Val Boc-Cpa Boc-T36 Example 29 none 218 Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T39 I Example 29 none 219 Bts-D-Phe(3Cl) Boc-D-Val Boo-Nva Boc-T37 Example 29 none 220 Bts-D-Phe(3Cl) Boc-D-Val Boc-Nva Boc-T39 Example 29 none 221 Bts-D-Phe(3Cl Boc-D-Val Boc-Nva Boc-T35 Example 29 none 222 Bts-D-Tyr(3tBu) Boc-D-Val Boc-Nva Boc-T9 Example 29 none 223 Bts-D-Tyr(But) Boc-D-Val Boc-Nva Boc-T9 Example 29 acetylation 224 Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 reductive amination with formaldehyde 225 Bts-D-1-Nal Boc-D-val Boc-Leu Boc-T9 Example 29 acetylation 226 Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 reductive amination with aldehyde 227 Bts-D-1-Nal Boc-D-Val Boc-Leu Boc-T9 Example 29 reductive amination with benzaldehyde Notes Any amino acid or tether designated as the Boc derivative could be substituted with the corresponding Ddz derivative. - (Compounds 2, 5, 6, 8, 9, 56 and 65 are not compounds of the invention).
1H and 13C NMR spectra were recorded on a Varian Mercury 300 MHz spectrometer and are referenced internally with respect to the residual proton signals of the solvent. Information about the conformation of the molecules in solution can be determined utilizing appropriate two-dimensional NMR techniques known to those skilled in the art. HPLC purifications were run on a Waters XTerra MS C18 column, using the Waters FractionLynx system. Automated medium pressure chromatographic purifications were performed on an Isco CombiFlash 16x system with disposable silica or C18 cartridges that permitted up to sixteen (16) samples to be run simultaneously. MS spectra were recorded on a Waters Micromass Platform II or ZQ system. HRMS spectra were recorded with a VG Micromass ZAB-ZF spectrometer. Chemical and biological information were stored and analyzed utilizing the ActivityBase database software (IDBS, Guildford, Surrey, UK). - HPLC analyses are performed on a Waters Alliance system 2695 running at 1 mL/min using an Xterra MS C18 column 4.6 x 50 mm (3. 5 µm). A Waters 996 PDA provided UV data for purity assessment. An LCPackings splitter (50:40:10) allowed the flow to be separated in three parts. The first part (50%) went to a Micromass Platform II MS equipped with an APCI probe for identity confirmation. The second part (40%) went to an evaporative light scattering detector (ELSD, Polymer Laboratories, PL-ELS-1000) for purity assessment and the last portion (10%) to a chemiluminescence nitrogen detector (CLND, Antek Model 8060) for quantitatlon and purity assessment. Data was captured and processed utilising the most recent version of the Waters Millenium software package.
- An example LC method suitable for compounds of the present invention uses MeOH as solvent A, H2O as solvent B and 1%TFA/ H2O as solvent D. Initial mobile-phase composition is 5% A, 85% B and 10% D. Details of the standard gradient method are shown below:
Time A% B% D% Curve 0.00 5 85 10 6 1.00 5 85 10 6 6.00 50 40 10 6 9.00 50 40 10 6 14.00 90 0 10 6 17.00 90 0 10 6 17.50 5 85 10 6 20.00 5 85 10 6 - Compounds 2-8, 8-10, 56, 65 and 144 are as defined in Table (3), herelnbelow.
- Yield: 12 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300MHz, DMSO-d8) δ 8.83 (m,1H); 8.53 (m,1H); 7.83 (m, 1H); 7.4-7.08 (m, 7H); 7.00-8.84 (m, 2H); 6.80 (d, 15Hz. 1H); 6.41 (dt, 15Hz, 5.4 Hz, 1H); 4.35 (m, 1H); 4.25-4.05 (m, 3H); 3.94 (dt, 1H, 6Hz, 15Hz); 3.79 (dd, 1H, 3.6Hz, 8.4 Hz); 3.60 (m, 1H); 3.52-3.40 (bd, 1H); 3.22-3.06 (m, 4H); 1.88 (m, 2H); 1.54-1.28 (m, 2H); 1.25 (d, 3H, 4.8Hz); 1.22 (d, 3H, 2.7 Hz); 0.92-0.80 (m, 6H).
HRMS calc. for C40H40N4O4: 520.3049; found 520.3057 ± 0.0016
HPLC [standard gradient method (refers to that presented In General Methods for Analytical HPLC Analyses)] tR = 9.55 min. - Yield: 12 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d8) δ 9.35 (b, 1 H); 8.98 (b, 1 H); 5.62 (d, 1 H, 8.4Hz); 8.38 (b, 1H); 7.25 (b, 1H); 7.13-7.07 (m, 4H); 6.86 (t. 2H, 7.5Hz); 6.57 (d, 2H, 8.7 Hz); 4.33 (b,1,H); 4.21-4.02 (m, 3H); 3.78 (dd, 1H, 3.3Hz; 8.1Hz); 3.65-3.54 (m, 1H); 3.31-3.23 (m, 1H); 3.13-3.02 (m, 4H); 2.78-2.2.28-2.18 (m, 1H); 2.0-1.80 (m, 2H); 9.50-1.30 (m, 3H); 1.25 (d, 3H, 4.5Hz); 1.22 (d, 3H, 4.5Hz); 1.01 (d, 3H, 6.6Hz): 0.90 (d, 3H, 6.6Hz); (t, 3H, 7.5Hz).
13C NMR (75.5MHz, DMSO-d8) δ 172.22; 171.37; 157.77; 157.44;158.04; 131.76; 130.80; 130.70; 127.88; 121.82; 115.83; 111.71; 62.13; 60.62; 54.21; 52.81; 47.13; 42.47; 33.31; 29.69; 29.30; 28.61; 20.36; 19.44; 18.72; 17.60; 13.97.
HRMS calc. for C30H42N4O5: 538.3155; found: 538.3145 ± 0.0016
HPLC (standard gradient) tR = 8.12 min. - Yield; 17 mg pure macrocycle was obtained (CLND quantlfication).
1H NMR (300 MHz, DMSO-d6) δ 9.02 (b, 1H); 8.47 (d, 1H, 8.4Hz); 7.7 (b, 1H); 7.58 (d, 1H, 5.4Hz); 7.28 (dd, 1H, 7.8Hz, 0.8Hz); 7.20 (t, 1H, 9.0Hz, 0.8Hz); 7.14 (d, 2H, 8.4Hz); 6.98-6.91 (m, 3H); 6.66 (d, 8.7Hz); 6.63 (d, 1H, 15.0Hz); 6.43 (dt, 1H, 6.0Hz, 15.0Hz); 4.28-3.86 (m, 6H); 3.60-3.40 (m, 2H); 3.22-3.12 (m, 1H0; 3.05 (d, 2H, 5.4Hz); 1.92-1.80 (m, 1H); 1.56-1.40 (m, 1H); 1.36-1.20 (m, 2H); 1.25 (d, 3H, 6.6Hz); 0.84 (t, 3H, 7.2Hz).
13C NMR (75.5MHz, DMSO-d6) δ 172.54:171.86; 158.97; 158.56; 127.39; 155.84; 131.62; 129.73; 129.20; 129.02; 128.43; 126.30; 124.51; 122.01; 115.85; 112.88; 61.23; 52.90; 51.23; 47.08; 42.66; 36.13; 33.30; 21.14; 19.57; 17.07; 14.14; 11.49.
HRMS calc. for C28H36N4O8: 508.2685; found: 508.2681 ± 0.0015
HPLC (standard gradient) tR = 7.67 min. - Yield: 18 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300MHz, DMSO-d6) δ 9.37 (b, 1H); 8.87 (b, 1H); 8.61 (d, 1H, 8.7Hz); 7.62 (b, 1H); 7.27 (d, 1H, 7.8Hz); 7.21 (t, 1H, 8.4Hz); 7.14 (d, 2H, 8.4Hz); 6.98-6.87 (m, 3H); 6.64 (d, 2H, 8.1Hz); 6.70 (d, 1H,15.6Hz); 6.39 (dt, 1H, 6.3Hz. 15.6Hz); 4.44-4.36 (m, 1H); 4.34-4.08 (m, 2Hz); 4.45-3.92 (dt, 1H, 6.9Hz, 15.6hz); 3.74 (dd, 1H, 3.6Hz, 8.4Hz); 3.54-3.26 (m, 3H); 3.22-3.02 (m, 3H); 2.60-2.36 (m, 4H); 2.24-2.14 (m, 1H); 2.02 (s, 3H); 1.96-1.89 (m, 1H); 1.80-1.66 (m, 1H); 1.01 (d, 3H, 6.3Hz); 0.90 (d, 3H, 6.6Hz).
13C NMR (75.5MHz, DMSO-d6) δ 171.51; 171.26; 158.90; 158.49; 157.38; 155.86; 131.63; 129.82; 129.21; 128.86; 128.63; 126.21; 121.98; 115.83; 112.83; 62.11; 61, 08; 51.97; 47.10; 42.78; 30.91; 30.67; 29.34; 20.37; 19.39; 15.06.
HRMS calc. for C30H40O5S: 568.2719; found: 568.2711 ± 0.0017
HPLC Rt (general method) 7.92 min. - Yield: 27 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300MHz, DMSO-d6) δ 9.05 (b, 1H); 8.43 (b, 1H); 8.34 (d, 1H, 9.3Hz); 7.40 (b, 1H); 6.97 (d, 1H, 7.5Hz); 6.92.6.74 (m,9H); 6.67-6.54 (m, 2H); 6.33-6.25 (m, 3H); 6.10 (dt, 1H, 5.7Hz, 16.2Hz); 4.22 (dt, 1H, 0.9Hz, 12Hz); 3.94-8.88 (m, 4H); 3.30 (dd, 1H, 3.6Hz, 7.8Hz); 3.24 (m, 1H); 3.18 (m, 1H); 2.85-2.68 (m, 3H); 2.44-2.23 (m, 2H); 1.32 (o, 1H, 7.5Hz); 0.97-0.89 (m, 1H); 0.42 (d, 3H, 6.6Hz); 0.01 (d. 3H, 6.6Hz).
13C NMR (75.5MHz, DMSO-d6) 5 171.20; 157.35; 155.88; 139.12; 131.61; 130.87; 129.74; 129.21; 128.77; 128.88; 126.85; 126.19; 121.97; 115.82; 112.84; 62.04; 61.10; 55.07; 50.01; 47.09; 42.85; 37.42; 29.11.
HRMS calc. For C34H42N4O5: 586.3155; found: 586.3145 ± 0.0017
HPLC Rt (general method) 9.34 min. - Yield: 17 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300MHz, DMSO-d6) δ 9.39 (b,1H); 8.83 (b, 1H); 8.29 (d, 1H, 9.3Hz); 7.62 (b, 1H); 7.28 (d, 1H, 6.6Hz); 7.20 (t, 1H, 6.9Hz); 7.12 (d, 2H, 7.8Hz); 6.98-91 (m, 2H); 6.63 (d, 2H, 8.4Hz); 6.58 (d, 1H,18.2Hz); 6.40 (dt, 1H, 5.7Hz, 16.2Hz); 4.29-4.13 (m, 3H); 4.03-3.92 (m, 2H); 3.52 (m, 1H); 3.15-3.05 (m, 3H); 2.45-2.37 (m, 1H);
1.96-1.88 (m, 1 H); 1.25 (dd, 2H, 4.5Hz; 6Hz); 1.01 (d, 3H, 6.3Hz); 0.91 (d, 3H, 6,6Hz); 0.86 (d, 3H, 7.2Hz); 0.81 (d, 3H, 6.6Hz).
13C NMR (75.5MHz, DMSO-d6) δ 171.85; 171.17; 157.37; 155.87; 131.59; 129.88; 129.18; 128.97; 128.78; 128.51; 126.16; 121.97; 115.83; 112.85; 61.55; 61.18; 58.15; 54.22; 47.08; 42.89; 36.32; 29.35; 29.00; 20.34; 19.56; 18.73; 17.44.
HRMS calc. for C30H40N4O5 536.2998; found: 536.2990 ± 0.0017.
HPLC (standard gradient) tR = 8.15 min. - Yield: 24 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6) δ 9.33 (b, 1H); 8.82 (b, 1H); 8.56 (d, 1H, 8.3Hz); 7.60 (b, 1H); 7.27 (d, 2H, 7.8Hz); 7.20 (t, 1H, 7.8Hz); 7.13 (d, 2H, 8.4Hz); 6.95 (t, 2H, 7.8Hz); 6.64 (d, 2H, 8.4Hz): 6.57 (d, 1H, 15.4Hz); 6.38 (dt, 1H, 15.4Hz, 5.8Hz); 4.26-4.10 (m, 3H); 3.96 (dt, 1H, 5.4Hz, 8.4Hz); 3.77 (dd, 1H, 3.7Hz, 7.8Hz); 3.51-3.24 (m, 3H); 3.18-3.02 (m, 3H); 1.90 (h, 1H, 6.4Hz); 1.73-1.54 (m, 2H); 1.45 (dt, 1H, 6.7Hz. 0.9Hz); 0.99 (d, 3H, 6.6Hz); 0.89 (d, 3H, 6.3Hz); 0.87 (d, 3H, 6.0Hz); 0.80 (d, 3H, 6.3Hz).
13C NMR (75.5MHz, DMSO-d6) δ 172.23; 171.17; 157.37; 155.88; 131.62; 129.82; 129.19; 128.95;128.59; 126.24; 121.99; 115.84; 112.88; 64.23; 61.98; 61.14; 51.43; 61.14; 51.43; 47.07; 42.81; 29.38; 24.85; 24.11; 21.00; 20.32; 19.30.
HRMS calc. for C31H42N4O5 550.3155; found: 550.3150 ± 0.0016.
HPLC (standard gradient) tR = 8.91 min. - Yield: 16 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6) δ 9.39 (b, 1H); 8.90 (b, 1H); 8.67 (d, 1H, 8.4Hz); 7.74 (b, 4H); 7.29-7.08 (m, 4H); 6.99-6.87 (m, 2H); 6.64 (d, 2H, 8.1 Hz); 6.61 (d, 1H, 16.5Hz); 6.40 (dt, 1H, 5.7Hz, 16.5Hz); 4.40-4.06 (m, 4H); 4.02-3.95 (m, 1H); 3.79 (dd, 1H, 3.6Hz, 7.8Hz); 3.55-3.30 (m, 2H); 3.16-3.05 (m, 3H); 2.82-2.69 (m, 2H); 2.02-1.85 (m, 2H); 1.64-1.43 (m, 3H); 1.29-1.23 (m, 1H); 1.01 (d, 3H, 6.3Hz); 0.91 (d, 3H, 6.3Hz); 0.86-0.84 (m, 2H).
HPLC (standard gradient) tR = 5.71 min. - Yield, 17 mg pure macrocycle was obtained (CLND quantification).
1H NMR (300 MHz, DMSO-d6) δ 9.60 (b, 1H); 9.39 (b, 1H); 8.88 (b, 1H); 8.70 (d, 1H, 7.5Hz); 8.57 (d, 1 H, 4.2Hz); 7.27 (t, 6Hz); 6.96 (d, 2H, 8.4Hz); 6.66 (d, 2H, 8.4Hz); 5.78-5.68 (m, 1H); 5.42-5.33 (m,11H); 3.96-3.89 (m, 1H); 3.80-3.57 (m, 5H); 3.41-3.34 (m, 1H); 3.10-2.90 (m, 1H); 2.78-2.66 (m, 1 H); 2.21-2.10 (m, 1H); 2.06-1.93 (m, 1H); 1.70-1.60 (m, 1H);1.52-1.41 (m, 1H); 1.39-1.26 (m, 1H); 1.25 (d, 3H, 4.8Hz); 1.23 (d, 3H, 4.5Hz); 0.83 (dd, 3H, 3Hz, 4.5Hz).
13C NMR (75.5MHz, DMSO-d6) δ 172.68; 172.63; 159.15; 158.73; 157.38; 157.25; 130.89; 124.99; 116.03; 62.51; 62.12; 54.29; 49.27; 42.47; 32.77; 30.43; 28.85; 20.46; 19.59; 18.72; 17.39; 13.90; 13.09.
HRMS calc. for C24H36N4O4: 444.2736; found: 444.2726 ± 0.0013
HPLC (standard gradient) tR = 6.80 min. - 1H NMR (300 MHz, CD3OD) δ 7.4 (m, 1H); 7.27 (dt, 1H, 1.5 Hz, 6.6 Hz); 7.22-7.14 (m, 2H); 7.08-6.98 (m, 2H); 6.78 9t, 2H, 6.6 Hz); 4.45-4.39 (m, 2H); 4.15 (d, 2H, 8.1 Hz); 7.74 (d, 1H, 9.3 Hz); 3.54 (d, 1H, 10.8 Hz); 3.35-3.22 (m, 2H); 3.20 (q, 1H, 1.5 Hz); 2.82-2.71 (m, 1H); 2.61-2.55 (m, 1H); 2.21-2.11 (m, 1h); 2.02-1.94 (m, 1H); 1.74-1.40 (m, 5H); 1.04 (d, 3H, 6.6 Hz); 0.93 (d, 3H, 6.6 Hz); 0.74-0.64 9m, 1 H); 0.45-0.28 (m, 2H); 0.15-0.08 (m, 1 H); 0.06-0.02 (m, 1 H).
- 13C NMR (75.5 MHz, CD3OD) δ 173.29; 172.14; 167.51; 155.47; 134.86; 134.81; 130.38; 130.31; 128.81; 128.25; 127.44; 121.63; 110.39; 107.71; 105.02; 67.10; 66.66; 62.81; 62.06; 60.10; 53.99; 41.44; 36.07; 31.91; 30.01; 29.18; 28.94; 27.79; 23.68; 23.15; 19.08; 18.25; 8.17; 4.98; 3.16.
HRMS: calc. for C31H41N4O4Cl 568.2816; found 568.2802 ± 0.0017 -
Table 2: Analysis of selected compounds of the invention (Compounds 1, 2, 5-12, 20, 21, 34, 38-41, 52, 55-60, 65, 71, 72, 76, 77, 80, 85, 98, 99, 120, 121, 127, 134, 177, 194, 197 and 199-202 are not compounds of the invention). Molecular Formula Molecular Weight (calculated) Monoisotopic Mass M+H Found 1 C30H40N4O5 536.7 536 537 2 C30H40N4O4 520.7 520 521 3 C30H42N4O4 522.7 522 523 4 C30H42N4O5 538.7 538 539 5 C28H36N4O5 508.6 508 509 6 C30H40N4O5S 568.7 568 569 7 C31H42N4O5 550.7 550 551 8 C34H42N4O5 566.7 586 587 9 C30H40N4O5 536.7 536 537 10 C31H42N4O5 550.7 550 551 11 C34H44N4O4 572.7 572 573 12 C29H38N4O5 622.6 522 523 13 C31H44N4O4 536.7 536 537 14 C35H46N4O4 586.8 586 587 15 C30H41N4O4Cl 557.1 556 557 16 C30H41N4O4Cl 557.1 556 557 17 C32H43N5O4 561.7 561 562 18 C29H40N4O5 524.7 524 525 19 C30H41N4O4F 540.7 540 541 20 C31H42N4O4 534.7 534 535 21 C36H44N4O4 584.7 584 585 22 C31H44N4O5 552.7 552 553 23 C34H44N4O4 572.7 572 573 24 C28H40N4O4S 528.7 528 529 25 C30H41N4O4Cl 657.1 556 557 26 C31H421d4O5 550.7 550 551 27 C27H39N5O4S 529.7 529 530 28 C29H41N5O4 523.7 523 524 29 C28H39N5O5 525.6 525 526 30 C30H41N3O6 539.7 539 540 34 C34H40N4O6 600.7 600 601 38 C28H36N4O5 508.6 508 509 39 C28H38N4O5 508.6 508 509 40 C27H34N4O5 494.6 494 495 41 C34H40N4O5 584.7 584 585 62 C33H38N4O5 570.7 570 571 55 C31H43N5O5 565.7 565 566 56 C30H41N5O5 551.7 551 552 57 C28H36N4O6 524.6 524 525 68 C34H40N4O6 600.7 600 601 59 C36H41N5O5 623.7 623 624 60 C35H42N4O6 614.7 614 615 65 C24H36N4O4 444.6 444 4.45 71 C29H40N4O6 540.7 540 541 72 C38H42N4O5 634.8 634 635 76 C38H42N4O5 634.8 634 635 77 C31H42N4O5 550.7 550 551 80 C31H42N4O5 550.7 550 551 86 C30H40N4O5 536.7 536 537 87 C36H46N4O4 598.8 598 599 88 C34H50N4O5 594.8 594 595 89 C31H44N4O4 536.7 536 537 90 C36H46N4O4 598.8 598 599 91 C30H42N4O5 538.7 538 539 92 C31H44N4O5 552.7 552 553 96 C28H38N4O5 510.6 510 511 97 C33H46N4O5 578.7 578 579 98 C24H39N5O4 461.6 461 462 99 C24H39N5O4 461.6 461 462 109 C29H41N5O5 536.7 539 540 110 C29H41N5O5 539.7 539 540 111 C30H41N3O6 539.7 539 540 112 C31H44N4O5 552.7 552 553 120 C30H38N4O5 534.6 534 535 121 C32H45N5O8 595.7 595 596 122 C31H43N4O4Cl 571.2 570 571 123 C29H41N5O4 523.7 523 524 124 C29H41N5O4 523.7 523 524 126 C30H40N4O5 538.7 536 537 128 C32H46N4O5 566.7 566 567 127 C30H38N6O3S 562.7 562 563 128 C32H46N4O5 566.7 566 567 129 C35H46N4O4 586.8 586 587 130 C29H42N4O4S 542.7 542 543 131 C31H43N4O4Cl 571.2 570 571 132 C31H43N4O4Cl 571.2 570 571 133 C31H43N4O4F 554.7 554 555 134 C25H37N4O3Cl 477.0 476 477 135 C31H45N5O5 567.7 567 568 136 C34H45N5O4 587.8 587 588 137 C28H41N5O4S 543.7 543 544 138 C30H42N5O4Cl 572.1 571 572 139 C30H42N5O4Cl 572.1 571 572 140 C30H42N5O4F 555.7 555 556 141 C32H44N4O5 564.7 564 565 142 C35H44N4O4 584.7 584 585 143 C29H40N4O4S 540.7 540 541 144 *C31H41N4O4Cl 569.1 568 569 145 C31H41N4O4Cl 569.1 568 569 148 C31H41N4O4F 552.7 552 553 147 C31H43N5O5 565.7 565 566 148 C34H43N5O4 585.7 585 586 149 C30H40N5O4Cl 570.1 569 570 150 C30H40N5O4Cl 570.1 589 570 151 C30H40N5O4F 553.7 553 554 152 C29H41N5O5 539.7 539 540 153 C32H41N5O4 559.7 559 560 154 C26H37N5O4 515.7 515 516 155 C28H38N5O4Cl 544.1 543 544 156 C28H38N5O4Cl 544.1 543 544 157 C28H38N5O4F 527.6 527 528 158 C27H37N6O4Cl 545.1 544 545 159 C31H44N4O5 552.7 552 553 160 C31H44N4O6 552.7 552 553 161 C31H45N5O4 551.7 551 552 162 C31H44N4O4 536.7 538 537 163 C31H44N4O4 536.7 536 537 164 C31H44N4O4 536.7 536 537 165 CC31H44N4O5 552.7 552 553 166 C31H44N4O5 552.7 552 553 167 C32H42N4O4S 578.8 578 579 168 C28H40N4O4S 528.7 528 529 169 C31H43N4O4Cl 571.2 570 571 170 C30H40N4O4Cl2 591.6 590 591 171 C30H40N4O4F2 558.7 558 559 172 C32H46N4O6 582.7 582 583 173 C34H43N3O5 573.7 573 574 174 C31H43N3O6 553.7 553 554 175 C31H44N4O5 552.7 552 553 176 C31H44N4O5 552.7 552 553 177 C29H40N4O5 524.7 524 525 178 C29H40N4O6 540.7 540 541 179 C32H40N4O5 560.7 560 561 180 C26H36N4O5S 516.7 516 517 181 C28H37N4O5Cl 545.1 544 545 182 C28H37N4O5Cl 545.1 544 545 183 C28H37N4O5F 528.6 528 529 184 C31H40N6O4 560.7 560 561 185 C27H37N6O4Cl 545.1 544 545 186 C31H40N6O5 576.7 576 577 187 C31H41N4O4F3 590.7 590 591 188 C30H41N4O4F 540.7 540 541 189 C30H41N5O6 567.7 567 568 190 C33H42N4O4S 590.8 590 591 181 C32H44N4O5 564.7 564 565 192 C31H40N4O4Cl2 603.6 602 603 193 C31H40N4O4F2 570.7 570 571 194 C32H48N6O6 612.8 612 613 195 C32H46N4O5 566.7 566 567 198 C32H43N6O4Cl 611.2 610 611 197 C32H45N8O5Cl 629.2 628 629 198 C32H43N4O4Cl 583.2 582 583 199 C27H39N4O6Cl 551.1 550 551 200 C31H39N4O4Cl 567.1 566 567 201 C34H42N4O4 570.7 570 571 202 C31H42N4O5 550.7 550 551 203 C30H40N5O5Cl 686.1 585 586 204 G29H40N7O4Cl 586.1 585 586 205 C32H45N4O4Cl 585.2 584 585 206 C29H40N5O6SCl 622.2 621 622 207 C29H39N6O5Cl 587.1 586 587 208 C29H41N7O5 567.7 567 568 209 C30H41N5O6 567.7 567 568 210 C31H45N5O5 567.7 567 568 211 C30H42N5O4Cl 572.1 571 572 212 C31H44N5O4Cl 586.2 585 586 213 C30H40N4O512 790.5 790 791 214 C30H42N4O6 554.7 554 555 215 C30H43N5O5 553.7 553 554 216 C32H43N4O4Cl 583.2 582 583 217 C31H40N4O4FCl 587.1 588 587 218 C31H43N4O4Cl 571.2 570 571 219 C30H40N4O4Cl2 591.6 590 591 220 C31H43N4O4F 554.7 554 555 221 C30H40N4O4FCl 575.1 574 575 222 C34H50N4O5 594.8 594 595 223 C32H44N4O6 580.7 580 581 224 C36H48N4O4 600.8 600 601 225 C37H48N4O5 628.8 628 629 226 C39H49N5O4S 683.9 683 684 227 C42H52N4O4 676.9 676 677 Notes 1. Molecular formulas and molecular weights (MW) are calculated automatically from the structure via ActivityBase software (IDBS, Guildford, Surrey, UK) or, for MW only, from the freeware program Molecular Weight Calculator v. 6.32 2. M+H obtained from LC-MS analysis using the General Method as described 3. All analyses conducted on material after preparative HPLC purification - The compounds described herein were evaluated for their ability to interact at the human motilin receptor utilizing a competitive radioligand binding assay as described in Method B1. Further characterization of the interaction can be performed utilizing the functional assays described in Methods B2, B3 and B4. Some of these methods can be conducted, if so desired, in a high throughput manner to permit the simultaneous evaluation of many compounds. Other assays have also been described that are suitable for HTS, such as that based upon the stable expression of a synthetic gene for the human motilin receptor.
- Results for the examination of representative compounds of the present invention using Method B1 are presented in Table 3, compounds 1, 2, 5-12, 20, 21, 34, 38-41, 52, 55-60, 65, 71, 72, 76, 77, 80, 85, 98, 99, 127, 134, 177, 194, 197 and 199-202 not being compounds of the invention. The binding activity is listed as ranges with the following levels: A = 0.001-0.10 µM; B = 0.10-1.0 µM; C = 1.0-10.0 µM. In addition, the assay results of two additional compounds using this Method are shown below. As can be observed, this demonstrates the activity of a representative bicyclic compound of Formula IV, which resulted from incorporation of D-proline as the second recognition building block. Significantly, the lack of binding activity obtained with compound 121, which is the linear analogue of compound 1 (Ki = level B), illustrates the critical importance of the cyclic structure to attaining the desired interaction.
-
- For determination of functional significance of the binding, the compounds are preferably tested in the Aequorin assay as described in Method B2, although the procedure of Method B3 is also applicable. As can be seen from the data presented in Table 4, the representative compounds examined act as antagonists at the motilin receptor and are devoid of agonist activity at the concentrations studied. The functional activity is listed as ranges with the following levels: A = 0. 001-0.10 µM; B = 0.10-1.0 µM. The higher sensitivity of the assay of Method B2, almost 100 times that of Method C, makes it the preferred one for this assessment. This is evident in the EC50 values obtained in each for the positive agonist standard, motilin. Additionally, Method B2 measures the actual signaling event, which makes It more relevant to the effect that is desired, whereas the assay of Method B3 simply measures GTP turnover.
Table 4: Demonstration of Antagonist Activity at the Motilin Receptor Aequorin (Method B2)1 Compound Binding (Ki) IC50 142 A B 149 A B 167 A A 168 A A 212 A A Motllin (human, porcine)2 0.6 not applicable 1 Activity is listed as ranges with the following levels: A = 0.001-0.10 µM; B = 0.10-1.0 µM
2Human and porcine motllin are the same peptide. - In addition, a common and scientifically-accepted ex vivo assay for the measurement of agonist or antagonist activity at the motilin receptor is the contraction of rabbit duodenum or other gastrointestinal smooth muscle tissue. A2-A4 Agonists are defined as compounds that induce >50% contraction relative to the motilin peptide, whereas antagonists are defined as compounds that cause >50% inhibition of the response to motilln. Compounds of the present invention have shown significant antagonist activity in this assay. For example, compound 144 exhibited a pA2 = 6.95, while compound 165 had a pA2 = 7.17, as calculated from the Schild plots of the response obtained at various concentrations as described In Method B4.
- Gastric motility Is generally measured In the clinical setting as the time required for gastric emptying and subsequent transit time through the Gl tract. Gastric emptying scans are well known to those skilled In the art an, briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solid and liquids can be measured independently.
- A test food or liquid is radiolabeled with an isotope (88mTc) and after ingestion or administration, transit time through the Gl tract and gastric emptying are measured by visualization using gamma cameras. These studies are performed before and after the administration of the therapeutic agent to quantify the efficacy of the compound.
-
- Membranes were prepared from CHO cells stably transfected with the human motilin receptor and utilized at a quantity of 1.5 µg/assay point. [PerkinElmer™ SignalScreen Product #611054]
- [261I]-Motilin (PerkinElmer, #NEX-378); final concentration: 0.04-0.06 nM
- Matilin (Bachem™, #H-4385); final concentration: 1 µM
- Muldscreen Harvest plates-GF/B (Millipore™, #MAHFB1H60)
- Deep-well polypropylene titer plate (Beckman Coulter™, #267006)
- TopSeal-A (PerklnElmer, #6005185)
- Bottom seal (Millipore, #MATAH0P00)
- MicroScint-0 (PerkinElmer, #6013611)
- Binding Buffer: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA, 0.1% BSA
-
- 150 of membranes diluted In binding buffer
- 10 µL of compound diluted in binding buffer
- 10 µL of radioligand ([125I)-Motilin) diluted in binding buffer
- Final Test Concentrations (N=11) for Compounds:
- 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 µM.
- Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at -20°C until the day of testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.5%.
- In deep-well plates, diluted cell membranes (1.5 µg/mL) are combined with 10 µL of either binding buffer (total binding, N=5), 1 µM motilin (non-specific binding, N=3) or the appropriate concentration of test compound. The reaction is initiated by addition of 10 µl of [126I]-motilin (final conc. 0.04 - 0.06 nM) to each well. Plates are sealed with TopSeal-A, vortexed gently and incubated at room temperature for 2 hours. The reaction is arrested by filtering samples through pre-soaked (0.3% polyethyleneimine, 2 h) Multiscreen Harvest plates using a Tomtec Harvester, washed 9 times with 500 µL of cold 50 mM Tris-HCl (pH 7.4), and than plates are air-dried in a fumehood for 30 minutes. A bottom seal Is applied to the plates prior to the addition of 25 µL of MicroScint-0 to each well. Plates are than sealed with TopSeal-A and counted for 30 sec per well on a TopCount Microplate Scintillation and Luminescence Counter (PerkinElmer) where results are expressed as counts per minute (cpm).
- Data are analyzed by GraphPad™ Prism (GraphPad Software, San Diego, CA) using a variable slope non-linear regression analysis. Kl values were calculated using a Kd value of 0.16 nM for [126I]-motilin (previously determined during membrane characterization).
-
- Membranes were prepared using AequoScreen™ (EUROSCREEN, Belgium) cell lines expressing the human motilin receptor (cell line ES-380-A; receptor accession #AF034632). This cell line is constructed by transaction of the human motilin receptor Into CHO-K1 cells co-expressing Gα16 and the mitochondrially targeted Aequorin (Ref #ES-WT-A5).
- Motilin (Bachem, #H-4385)
- Assay buffer DMEM-F12 (Dulbeccoe's Modified Eagles Medium) with 15 mM HEPES and 0.1% BSA (pH 7.0)
- Coelenterazine (Molecular Probes™, Leiden, The Netherlands)
- Final Test Concentrations (N=5) for Compounds:
- 10, 3.16, 1, 0.316, 0.1 µM.
- Compounds were provided as dry films at a quantity of approximately 1.2 µmol, in pre-formatted 96-well plates. Compounds were dissolved in 100% DMSO at a concentration of 10 mM and stored at -20°C until further use. Daughter plates were prepared at a concentration of 500 µM In 30% DMSO with 0.1% BSA and stored at -20°C until testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration In the assay was 0.6%.
- Cells are collected from culture plates with Ca2+ and Mg2+-free phosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelleted for 2 minutes at 1000 x g, resuspended in assay buffer (see above) at a density of 5 x 106 cells/mL and incubated overnight in the presence of 5 µM coelenterazine. After loading, cells were diluted with assay buffer to a concentration of 5'x105 cells/mL.
- For agonist testing, 50 µl of the cell suspension was mixed with 50 µl of the appropriate concentration of test compound or motilin (reference agonist) in 96-well plates (duplicate samples). The emission of light resulting from receptor activation was recorded using the Functional Drug Screening System 6000 'FDSS 6000' (Hamamatsu Photonics K.K., Japan).
- For antagonist testing, an approximate EC80 concentration of motilin (i.e. 0.5 nM; 100 µL) was injected onto the cell suspension containing the test compounds (duplicate samples) 15-30 minutes after the end of agonist testing and the consequent emission of light resulting from receptor activation was measured as described in the paragraph above.
- Results are expressed as Relative Light Units (RLU). Concentration response curves were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA) by non-linear regression analysis (sigmoldal dose-response) based on the equation E=Emax/(1+EC50/C)n where E is the measured RLU value at a given agonist concentration (C), Emax Is the maximal response, EC50 is the concentration producing 50% stimulation and n is the slope index. For agonist testing, results for each concentration of test compound were expressed as percent activation relative to the signal Induced by motilin at a concentration equal to the EC80 (i.e. 0.5 nM). For antagonist testing, results for each concentration of test compound were expressed as percent inhibition relative to the signal Induced by motilin at a concentration equal to the EC80 (i.e. 0.5 nM).
-
- Membranes were prepared from CHO cells stably transfected with the human motilin receptor and utilized at a quantity of 1.5 µg/assay point. [PerkinElmer SlgnalScreen Product #6110544]
- GTPγS (Sigma, #G-8634)
- [35S]-GTPγS (PerkinElmer, #NEX-030H)
- Motilin (Bachem, #H-4385)
- 96-well FlashPlate microplates (PericinElmer, #SMP200)
- Deep-well polypropylene titer plate (Beckman Coulter, #267006)
- TopSeal-A (PerkinElmer, #6005185)
- Assay Buffer: 50 mM Tris (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1 µM GDP. 0.1% BSA
-
- 25 µL of compound diluted In assay buffer
- 25 µL of assay buffer (agonist assay) or 0.6 µM motilin (0.1 µM final concentration) diluted in assay buffer (antagonist assay)
- 100 µL of [35S]-GTPγS diluted In assay buffer
- Final Test Concentrations (N=12) for Compounds:
- 50, 20,10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 µM.
- Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at -20°C until the day of testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.5%.
- CHO membranes were immobilized into 96-well FlashPlate microplates. Test compound, GTPγS, motilin and [35S]-GTPγS were combined In each well according to the Assay Volumes described above.
- For the assay to measure agonist activity, an additional 25 µl of buffer was added to each well in addition to 25 µl of either buffer (basal value, N=4), 1 µM (final conc.) motilin (Emax value, N=3), 25 µM (final conc.) GTPγS (non-specific value, N=4), or the appropriate concentration of test compound (N=3).
- For the assay to measure antagonist activity, an additional 25 µL of either buffer (unstimulated control) or motilin (0.1 µM final conc.) Is added to each well, in addition to either 25 µL of buffer (basal value, N=3), 1 µM (final conc.) motilin (Emax value, N=3), 25 µM (final conc.) GTPγS (non-specific value, N=4), or the appropriate concentration of test compound (N=3).
- The reaction is heated by addition of 100 mL of [35S]-GTPγS to each well. Each plate Is sealed (TopSeal-A) and incubated in the dark at room temperature for 150 min. Then, plates are counted for 30 seconds per well on the TopCount NXT.
-
- Where Top and Bottom correspond to the top and bottom values of the dose-response curve calculated by GraphPad Prism).
- Duodenal segments were vertically suspended in organ chambers of 10 mL filled with Krebs buffer and connected to an isotonic force transducer, with a preload of 1 g. After a stabilization period, the muscle strips were challenged with 10-4 M acetylcholine and washed. This was repeated until a stable maximal contraption was obtained (2-3 times), with an Interval of at least 20 minutes.
- After a stable base line was reached, test compounds were added to the bath. After 15 min Incubation, a dose response to motilin was recorded by adding logarithmically increasing concentrations of motilin to the bath (final concentration 10-8 to 10-6 M). A blank experiment (no test compound present) was also performed. At the end of the dose response curve, a supramaximal dose of acetylcholine (10-4 M) was given and this response was used as a reference (100% contraction).
- The results of experiments at different concentrations of test compound were combined and analyzed to derive the pA2 value from the Schild plot.
-
- Radioligand competitive binding assays performed using Method B1
- Values reported as ranges: A = 0.001-0.100 µM; B = 0.100-1.0 µM; C= 1.0-10.0 µM
- X is NH except for:
-
-
-
-
Claims (15)
- The macrocyclic compound as claimed in claim wherein PG and PG' are each independently amine protecting groups selected from tert-butoxycarbonyl (Boc), 9-fluorenylmethoxy-carbonyl (Fmoc), α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl (Ddz) and allyloxycarbonyl (Alloc).
- The macrocyclic compound as claimed in claim 2 wherein the protecting group is Ddz.
- The macrocyclic compound as claimed in any one of claims 1 to 3 made from a compound of formula T33a or T33b, for example a compound of formula T33a.
- A process for preparing a conformationally-defined macrocyclic compound in accordance with the following scheme:BB1 are building blocks (amino acids, hydroxy acids) wherein BBi denotes in order from C- terminus in analogy to standard peptide nomenclature, not in order of addition;PG is a protecting group;P/AG are protecting group and/or activating group;Z is a reactive group; andPG-Y-TETHER-Z is a compound of formula T33a, T33b, T9, T38, T39, T40, T35, T36, T37, T11 or T32 as defined in any one of claims 1 to 4.
- A compound of any one of formulae T33a, T33b, T9, T38, T39, T40, T35, T36, T37, T11 or T32 as defined in claim 1.
- The compound as claimed in claim wherein PG and PG' are each independently amine protecting groups selected from tert-butoxycarbonyl (Boc), 9-fluorenylmethoxy-carbonyl (Fmoc), α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl (Ddz) and allyloxycarbonyl (Alloc).
- The compound as claimed in claim 8 wherein the protecting group is Ddz.
- The compound as claimed in any one of claims 7 to 9 wherein the compound is of formula T33a or T33b, for example wherein the compound is of formula T33a.
- A method of making a compound of formula T33a or T33b as defined in claim 4 comprising:(a) reducing a compound of formula 33-3aPG
- The method of claim 11 wherein:the compound of formula 33-3aPG is provided by effecting a Sonagashira reaction between propargylamine in which the amino group is protected with a group PG as defined in part (a) of claim 11 and a compound of formula 33-2a: or
- The method of claim 12 wherein the compound of formula 33-2a is provided by reduction of the corresponding ester, for example the methyl ester of formula 33-1a:
- The method of claim 13 wherein:the compound of formula 33-2a is made by reducing the methyl ester of formula (33-1a), which methyl ester is made by effecting a Mitsunobu reaction between 2-iodophenol and (S) -methyl lactate; orthe compound of formula 33-2b is made by reducing the methyl ester of formula (33-1b), which methyl ester is made by effecting a Mitsunobu reaction between 2-iodophenol and (R)-methyl lactate.
- The method of any one of claims 11 to 14, which is a method of making a compound of formula T33a.
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DK1633774T3 (en) | 2010-05-25 |
EP2210612A2 (en) | 2010-07-28 |
EP1633774B1 (en) | 2010-02-17 |
US7521420B2 (en) | 2009-04-21 |
US20050054562A1 (en) | 2005-03-10 |
CA2528375A1 (en) | 2004-12-23 |
US20120226072A1 (en) | 2012-09-06 |
US20090137835A1 (en) | 2009-05-28 |
US8497242B2 (en) | 2013-07-30 |
US8129561B2 (en) | 2012-03-06 |
WO2004111077A1 (en) | 2004-12-23 |
US20160221927A1 (en) | 2016-08-04 |
US20120226067A1 (en) | 2012-09-06 |
ATE457995T1 (en) | 2010-03-15 |
US20090240027A1 (en) | 2009-09-24 |
JP2007523853A (en) | 2007-08-23 |
US10040751B2 (en) | 2018-08-07 |
EP2210612A3 (en) | 2010-11-24 |
CA2528375C (en) | 2013-11-19 |
US20110245459A1 (en) | 2011-10-06 |
JP4928261B2 (en) | 2012-05-09 |
US20120165566A1 (en) | 2012-06-28 |
DE602004025569D1 (en) | 2010-04-01 |
US20120226066A1 (en) | 2012-09-06 |
EP1633774A1 (en) | 2006-03-15 |
US9181298B2 (en) | 2015-11-10 |
ES2338789T3 (en) | 2010-05-12 |
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